Phospholipid depot

ABSTRACT

The present invention is directed to compositions and methods of preparation of phospholipid depots that are injectable through a fine needle.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/523,860 filed Jun. 14, 2012, issued on Dec. 13, 2016 under U.S. Pat.No. 9,517,202 B2, which application is a continuation ofPCT/US2010/060964, filed Dec. 17, 2010, which application claimspriority to U.S. Provisional Patent Application No. 61/288,220, filedDec. 18, 2009, the teachings of all of which are hereby incorporated byreference in their entireties for all purposes.

FIELD OF THE INVENTION

The present invention relates to phospholipid depot compositions forinsulin and other drugs and methods for preparation.

BACKGROUND OF THE INVENTION

An injectable depot is designed to prolong the duration of action andreduced the frequency of injection for a drug. Such depots are generallyadministered by subcutaneous or intramuscular injection or by injectionor instillation into body tissues, vessels or cavities. A depot prolongsthe action of a pharmacologically active agent by releasing it intosurrounding tissues from a reservoir slowly over time. A 1-day, 7-day or30-day depot release profile, which enables a once-a-day, once-a-week oronce-a-month injection schedule, respectively, would be highly desirablefor convenience and better patient compliance.

Various materials have been employed for depot compositions. The mostcommon depot-forming materials are biodegradable synthetic polymers,e.g., polylactic-co-glycolic acid (PLGA) and polylactic acid (PLA). Thebiodegradable polymer depot generally comes in two common forms:microcapsules/microspheres and polymer gels. The PLGA/PLA depots havebeen used in several FDA approved drugs i.e., Zoladex™ (goserelinacetate) and Lupron Depot™ (leuprolide acetate), which are PLGAmicrocapsules and microsphere, respectively. Eligard™ is in a polymergel made by dissolving a drug and PLGA in a strong organic solvent i.e.,N-methyl-2-pyrrolidone.

A major disadvantage of the polymer depots is that they require largediameter needles for injection or implantation due to the physical sizeof the microcapsules/microspheres and/or the high viscosity of thepolymer gel. For example, 14- or 16-gauge (G) needles are required forimplantation of Zoladex™ and 18 G or 20 G needles for injection ofEligard™. However, in common medical practice, needles of size greaterthan 21 G are generally not used for injection because they causesignificant pain and psychological trauma for patients. For drugs likeinsulin, which are self-administered daily, fine 25-27 G needles and 1cc syringes are used. The injectability or ease with which the end usercan self-inject through such a system will be key to such a drug's usercompliance and therapeutic efficacy. For discussion purposes herein, theinjectability of a syringe-administered depot is quantitatively definedas meeting the “Acceptable Injectability Criterion” if it requires anapplied force of no more than 10 pounds to be extruded from a 1 ccsyringe through a 25 G ½ inch long needle at rate of 2 cc/min. Such ascenario represents typical conditions during the self-administration ofinsulin and other self-injected drugs.

Moreover, PLGA and PLA are insoluble in water and both require extremelystrong organic solvents such as methylene chloride, chloroform orN-methyl-2-pyrrolidone to fabricate the microcapsules/microspheres orgels. Unfortunately, most biological molecules such as protein drugs areincompatible with strong solvents. Methylene chloride orN-methyl-2-pyrrolidone, which are used in PLGA/PLA production, denatureinsulin immediately upon contact.

Phospholipids (PL) are naturally occurring substances in the human bodyand are the major constituents of cell membranes. These molecules havean established record of safety and biocompatibility as components ininjected medicines. PL are also generally insoluble in water (like thePLGA polymers) and following injection into tissue and coming intocontact with aqueous body fluids and tissues, PL can precipitate andtrap a co-administered drug, to form a drug-PL co-precipitate that canfunction as a depot. Over time, this mass diffuses slowly into asurrounding tissue and/or is degraded by phospholipase, which is anenzyme distributed throughout the body that slowly hydrolyzesphospholipids, resulting in a slow release of the trapped drug. Withsuch favorable safety, solubility and biocompatibility properties, itwould appear that phospholipids are ideal depot materials. However, todate, there has been few successful depot drug product based onphospholipids. One primary problem is the poor injectability associatedwith phospholipid-based compositions.

This inventor has discovered that a high concentration (i.e., 20-80%) ofphospholipids is generally required in order to form the mass thatpermits depot functionality. However, once the phospholipidconcentration exceeds about 20% in a composition, the compositionbecomes thick, viscous and difficult to inject through fine needleswithout using an excessively high force. For example, Phosal 50PG,Phosal 50SA, and Phosal 50MCT (produced by the America Lecithin Company)are liposome-forming compositions containing about 50% phospholipidsdissolved in propylene glycol/ethanol, oil, and medium chain oil,respectively. With their honey-like consistency, the Phosal compositionsare very difficult to inject using a conventional hypodermic needle andsyringe. It requires more than 20 pounds of force to extrude Phosalthrough a 25 G V2 inch long needle from a 1 cc syringe at a plungerspeed of 2 cc/min. Thus, it will take 2-5 minutes or more to manuallyextrude 1 mL of the Phosal-based depot through a 26 G needle even usinga very high force—which is impractical for general medical use anddefinitely not suitable for self-administration. Therefore, acceptableinjectability using fine hyperdermic needles has been a main reasonpreventing phospholipids from becoming useful depot materials. Thisinvention discloses phospholipid depots with surprisingly goodinjectability that meets the Acceptable Injectability Criterion, asdefined above.

Another difficulty working with phospholipids is that phospholipids areonly soluble in certain organic solvents (e.g., ethanol) or oil (e.g.,vegetable oil) and many drugs (such as insulin or other protein drugs)are only soluble and stable in water, but not soluble or stable insolvents or oils that can dissolve phospholipids. Therefore, it has beenimpossible to manufacture phospholipid-based depots using conventionalsolvent methods or other methods disclosed in prior art without havingthe solvent-sensitive drugs precipitate or degrade (See WO 2006/002050,U.S. Pat. No. 5,807,573, WO/1994/008623, U.S. Pat. No. 5,004,611 andHarry Tiemesseen, et al. (2004) European Journal of Pharmaceutics andBiopharmaceutics Volume 58 (2005), pp 587-593).

Another hurdle in the production of phospholipid depots relates todifficulty in preparing a depot suitable for injection under sterileconditions. Many drugs are heat-sensitive and cannot survive heatsterilization (e.g., autoclaving) or radiation sterilization. This isespecially true for biological drugs such as insulin and other proteindrugs. In many cases, the only practical way to sterilize aprotein-containing composition is by filtration through a 0.2- or0.45-micron pore membrane to remove any microbial contaminants. With a20-80% phospholipid content, the thick consistency of the depotcompositions precludes any possibility of sterilization by filtration.Therefore, this invention also teaches unique methods for preparingdepots that may be sterilized by filtration.

Insulin is the mainstay for treatment of virtually all type 1 and manytype 2 diabetic patients. Insulins and insulin formulations are dividedinto two types: (1) quick onset/short acting and (2) long-acting. Thefirst type (“preprandial”) is used to control transient elevated bloodglucose levels that occur after meals. Long-acting insulin is used tomaintain a controlled baseline level of glucose level over a longduration such as 12-24 hours. A long-acting insulin or insulinformulation is thus referred to as “basal insulin”

Basal insulin therapy is utilized to achieve “glycemic control,” whichis the maintenance of blood glucose levels at a constant and acceptablelevel without fluctuations. Sufficient glycemic control requires plasmaglucose levels to be maintained within normal limits (70-130 mg/dl, or3.9-7.2 mmol/L) and indistinguishable from that in a non-diabeticperson. Glucose level fluctuations, especially the high peaks andvalleys resulting from poor glycemic control, are high risk factors fordiabetes-associated complications that can lead to morbidity andmortality. Therefore, to achieve adequate glycemic control, an idealbasal insulin formulation should deliver insulin to the circulation at aconstant rate (i.e., peak-less) over a prolonged period of time, such as24 hours. Human insulin itself has a rapid onset and short duration ofaction (the half-life of insulin is only about 5-6 minutes in thecirculation). Therefore, a human insulin depot formulation requires anapproach that is capable of both sequestering and releasing it slowlyand constantly to address the requirements needed for a successful basalinsulin therapy.

The pharmacological efficacy of insulin can be readily monitored byfollowing the post-administration plasma glucose concentration-timeprofile and the plasma insulin concentration-time profile. The formermeasures insulin's glucose-lowering efficacy or the pharmacodynamic orPD profile and the latter measures the insulin plasma levels as apharmacokinetic or PK profile.

The currently available basal human insulin formulations in the USinclude the NPH (Neutral Protamine Hagedorn) insulin sold under thetrade names of Humulin N and Novolin N by Eli Lilly and Company and NovoNordisk, respectively. NPH insulin, which was invented in the 1930's byHans Christian Hagedorn, is a suspension of zinc-insulin crystallinecomplexes combined with the positively charged polypeptide, protamine.The complexation with zinc and protamine turns the insulin intoinsoluble particles after injection that slowly release insulin.

Despite its long history of use (over 70 years), NPH is not an idealdepot formulation for basal insulin therapy. The following shortcomingsare well known:

-   -   High C_(max): The NPH PK profile has a pronounced peak or        C_(min). that occurs in about 4 hours after subcutaneous        injection. This high C_(max) causes hypoglcermia. Since basal        insulin is typically given at bedtime, the 4 hr post-injection        hypoglycemic phase normally occurs when the patient is asleep.        However, if the patient were to awaken in the middle of the        night and get out of bed, the hypoglycemic episode could lead to        fainting.    -   Short duration of action: NPH releases a substantial amount of        its insulin within the first few hours and is depleted in about        14-16 hours, making it suitable only as a twice-a-day (BID)        formulation. This deficiency disqualifies NPH as a true,        once-a-day (QD) formulation.    -   High peak-to-valley ratio of plasma insulin: In clinical        practice, BID regimens for NPH are still unable to stem high        C_(max) (peak) and low C_(min) (valley) fluctuations. The        resulting sub-optimal glycemic control increases the risk for        diabetic complications.    -   Poor dose uniformity: For suspensions like NPH, an intrinsic        problem is the inability to achieve uniform        injection-to-injection dosing in a small volumes—even with        strict adherence to the rigorous pre-injection mixing/shaking        instructions. For NPH this difficulty is further compounded        because it is typically injected in very small volumes (<1 mL).        Thus, the variability with respect to the amount of insulin        injected dose-to-dose for NPH can be as high as 10-20%, which        also contributes to poor glycemic control.

More recently, two basal insulin drugs, LANTUS® (insulin glargine,Sanofi-aventis) and LEVEMIR® (insulin detemir, Novo Nordisk), weredeveloped and subsequently approved. Both LANTUS® and LEVEMIR® areinsulin analogs, in that they are chemically modified insulin and arenot the authentic human insulin molecule. In contrast to NPH, LANTUS®releases insulin in a “peak-less” (peak to trough ratio less than 5within 24 hours after each injection) PK profile over 24 hours, whichare key factors underlying the drug's applicability as a once-a-day doseand its achievement of better glycemic control. Compared to NPH,LEVEMIR® has a less spiky PK profile but its duration of action issomewhat similar to NPH, making it suitable only for BID dosing. Ofthese two basal insulin analogs, LANTUS® has clear advantages over NPHowing to its 24 hr peak-less insulin PK profile.

Recently, LANTUS® has been reportedly linked to certain cancers. The FDAnoted: “3 of 4 observational studies suggest an increased risk forcancer associated with use of Lantus.” (Pink Sheet, Jul. 6, 2009, p.30). LANTUS® is also associated with a high incidence of injection sitepain possibly due to its low pH formulation (pH 4). Unlike humaninsulin, the long-term safety of the insulin analogs are unclear.

Despite the recent advances for insulin drugs, there is a need forimproved basal insulin formulations that provide a 24 hr peak-less PKprofile. Moreover, there remains a need for a phospholipid depotsuitable for injection under sterile conditions. A method is needed toenable a water-soluble or solvent-incompatible drug to be incorporatedinto a phospholipid depot. The present invention satisfies these andother needs.

BRIEF SUMMARY OF THE INVENTION

The present invention provides compositions and methods for preparingphospholipid depots that are injectable through a fine needle.Advantageously, the gels are easily injectable through a fine needleeven though they preferably have a high phospholipid content (e.g.,20-80%). The inventive gels are substantially uniform or one-phase,i.e., the pharmacologically active agent is uniformly distributed andremains uniformly distributed throughout the gel matrix, even aftercentrifugation at 1000 RPM for 5 minutes. This invention also relates tounique methods for the preparation of depots that allow for the intimatemixing or incorporation of water-soluble or solvent-incompatible drugsinto phospholipid depots.

As such, in one embodiment, the present invention provides a one-phasegel composition, comprising:

20 to 80% by weight of one or more phospholipids;

optionally a pharmacologically active agent; and

0.1 to 70% by weight water, wherein the gel composition is extrudablethrough a 25 G ½ inch long needle from a 1 cc syringe at an extrusionrate of 2 cc/min by an applied force of no more than 12 pounds. Thephospholipid depots are one-phase gels that can be aqueous orsubstantially anhydrous. In preferred embodiments, the formulationcontains about 1 to about 20% pharmacologically active agent. In oneembodiment, the optional pharmaceutical active ingredient is absent ornot present and the gel is useful as a dermal filler.

In yet another embodiment, the present invention provides a method forpreparing a one-phase gel composition, comprising:

a) forming a primary dispersion comprising one or more phospholipid(s)and an excessive amount of water;

b) homogenizing the primary dispersion to form a nanodispersion with anaverage particle size of about 30 nm to about 200 nm in diameter;

c) optionally passing the nanodispersion through a 0.2- or 0.45-micronfilter; and

d) removing the excessive water to obtain a one-phase gel composition.

In certain embodiments, the one-phase gel is an aqueous gel. In otherembodiments, the one-phase gel is substantially an anhydrous gel. Incertain embodiments, the one-phase gel further comprises apharmacologically active agent. When a pharmacologically active agent ispresent, it may be is added before step “b” or it may be added afterstep “b.” In other embodiments, it may be added before as well as afterstep “b.”

In other embodiments, the present invention provides a one-phase aqueousgel made by methods herein. The gel can be aqueous or a substantiallyanhydrous gel made by methods herein.

In certain embodiments, the aqueous or substantially anhydrous gels aretransparent in appearance and silky smooth to the touch. Theologically,the inventive gels are shear thinning and thixotropic, which are desiredproperties for good extrudability/injectability through a fine needle.In contrast, the same compositions, when prepared by known prior artmethods, result in thick pastes that are very difficult or impossible toinject through a fine hypodermic needle.

These and other aspects, objects and embodiments will become moreapparent when read with the accompanying detailed description and thefigures that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show three translucent aqueous gels. FIG. 1A: aqueous gel(T-5) contains 37% water, but has no pharmacologically active agent inthe T-5 composition as in EXAMPLE 1. FIG. 1B and FIG. 1C: aqueous gelseach containing 100 unit/mL recombinant human insulin and 50% water butmade with soy lecithin (FIG. 1B) or a synthetic phospholipid (POPC)(FIG. 1C), both as in EXAMPLE 3.

FIG. 2 illustrates pharmacokinetic profile in dogs following asubcutaneous (SC) or intramuscular injection (IM) at 0.25 mg/kg dose ofbuprenorphine in an anhydrous gel (F-27 as in EXAMPLE 6).

FIG. 3 illustrates prolonged local analgesic/anesthetic efficacy oflidocaine in an anhydrous gel (F-20 as in EXAMPLE 8) in guinea pigsfollowing an intracutaneous injection compared to placebo anhydrous gel.

FIG. 4 shows difference in injectability through 27 G needles after5-second manual extrusion using 1 mL syringes between two phospholipidpreparations containing the same composition (T-4) as in EXAMPLE 1 butprepared by different methods. The clear gel is an anhydrous gel (in topsyringe) and was prepared according to the method in EXAMPLE 1 and theopaque gel (bottom syringe) was prepared by the method taught in otherprior arts wherein all components were mixed and homogenized. Afterapplying the same force and duration to the syringes, substantially moreaqueous gel was ejected, compared to the opaque paste prepared accordingto other existing methods.

FIG. 5 illustrates the superior uniformity and physical stability of anaqueous gel produced using the methods of the present invention (right,T-4 as in EXAMPLE 1) compared to a paste produced by mixing the samecomponents but using a different process than the aqueous gel (left).After centrifugation (13,000 rpm, 10 minutes), the paste separates intoliquid and solid phases whereas the aqueous gel remains as a uniform,single-phase gel.

FIGS. 6A-6B. show a representative injection force versus time profilefor the insulin-containing aqueous gel (F-43) in EXAMPLE 14 (upperpanel). The test measured the force necessary to eject the gel from a 1cc syringe through a 25 G ½ inch long needle at rate of 2 cc/min. Forcomparison, the force profile for glycerin is shown in the lower panel.With a maximum injection force of less than 1.25 pounds, F-43 can beregarded as very injectable. Even as a gel, it was much easier to injectthan the liquid glycerin, which required approximately 8 pounds offorce.

FIG. 7 shows blood glucose levels following a subcutaneous injection ofa 20 IU/kg insulin dose for four different basal insulin formulations inthe streptozotosin (STZ)-induced type-I diabetic Sprague Dawley ratanimal model. Data points are mean values from 3 rats and error barsrepresent the standard error of the mean.

FIGS. 8A-8B show the plasma insulin levels (upper panel) measured usinga human insulin ELISA kit and the blood glucose levels (lower panel)measured by a glucometer following subcutaneous injection of twodifferent basal insulin formulations in the streptozotosin (STZ)-inducedtype-I diabetic Sprague Dawley rats. Data points are mean values from 4rats and error bars represent the standard error of the mean.

FIG. 9 is a schematic representation of the speculated conversion from ananodispersion (left) to a PG (right) upon removal of water. The circlesdepict the nanosized phospholipid particles in the nanodispersion, andthe space between the dots are filled with water as in an aqueous gel oroil as in an anhydrous gel.

FIG. 10 shows SAXS diffractograms for F-43 PG prepared according to themethod disclosed in the present invention (“F-43 PG according to thepresent invention”) and the same composition as F-43, but prepared bydirect mixing (“Same composition by other method”).

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The phrase “Acceptable Injectability Criterion” as used herein includesquantitatively defining a formulation that requires an applied force ofno more than 10 pounds to extrude the formulation from a 1 cc syringethrough a 25 G ½ inch long needle at rate of 2 cc/min. In certaininstances, the applied force is not more than 6 pounds, 7 pounds, 8pounds, 9 pounds, 10 pounds, 10.5 pounds, 11 pounds, 12 pounds, 13pounds, 14 pounds, 15 pounds, 16 pounds, 17 pounds, 18 pounds, 19 poundsor 20 pounds, to extrude the formulation from a 1 cc syringe through a25 G ¹/₂ inch long needle at rate of 2 cc/min. Such a scenariorepresents typical conditions during the self-administration of insulinand other self-injected drugs.

The term “acidifying agent” includes a pharmaceutically acceptable acidsuch as hydrochloric acid, acetic acid, and sulfuric acid, and the like.

As used herein, the term “alkalizing agent” includes a pharmaceuticallyacceptable base such as sodium hydroxide, potassium hydroxide, ammoniumhydroxide, lysine, arginine, and the like.

As used herein, the term “antimicrobial preservative” includes apharmaceutical additive that can be added to an injectablepharmacologically active agent and be used to inhibit the growth ofbacteria and fungi. The antimicrobial preservatives useful in thisinvention include, but are not limited to, cresols, phenol, benzylalcohol, ethanol, chlorobutanol, parabens, imidura, benzylkoniumchloride.

As used herein, the term “antioxidant” includes primarily reducingagents. The reducing agents useful in this invention include, but arenot limited to, ascorbic acid or salts thereof, ascorbyl palmitate,sodium metabisulfite, propyl gallate, butylated hydroxyanisole,butylated hydroxytoluene, tocopherol, methionine or salts thereof,citric acid or salts thereof, reducing sugars, or mixtures thereof.

As used herein, the term “aqueous phase” includes a water solutioncontaining pharmaceutically acceptable additives, such as acidifying,alkalizing, pH buffering, chelating, condensing and solubilizing agents,antioxidants and antimicrobial preservatives, tonicity/osmotic modifyingagent, other biocompatible materials or therapeutic agents. In certainembodiments, such additives assist in stabilizing the pharmacologicallyactive agent and depot compositions and in rendering the compositionsbiocompatible.

As used herein, the term “condensing agent” includes a pharmaceuticallyacceptable chemical that reduces solubility, alters release rate orincreases stability of the pharmacologically active agent. For example,zinc ion forms insoluble crystals of with insulin and causes the insulinto release slowly. Other examples may include aluminum ions, ferricions, protamine, or the like.

As used herein, the term “depot” includes a pharmacologically activeagent delivery composition that is capable for releasing thepharmacologically active agent in a slow or controlled manner into thesurrounding tissues to achieve a prolonged duration of action incomparison with the pharmacologically active agent without suchcomposition. A depot composition may be administered by injection,instillation, or implantation into soft tissues, a certain body cavityor occasionally into a blood vessel with injection through fine needlesbeing the preferred method of administration. A depot ofpharmacologically active agent is intended to provide (1) convenient orless frequent dosing, (2) prolonged action, (3) improved safety and/or(4) better drug efficacy. The term “depot composition” can be usedinterchangeably with “sustained-release composition,” “slow-releasecomposition,” “timed-release composition,” “extended-releasecomposition,” “delayed-release composition,” “long-acting composition,”or “controlled-release composition.”

As used herein, the term “emulsion” includes a mixture of immiscible oilphase and aqueous phase, where the oil phase comprises the oil andphospholipids and is in form of small droplets (the dispersed phase),which are suspended or dispersed in the aqueous phase (continuousphase). The primary emulsion formed in accordance with the presentinvention is typically optically opaque and possesses a finitestability.

As used herein, the term “a fine hypodermic needle” includes asmall-diameter, hollow needle that is used with a syringe to injectsubstances into the body. The outer diameter of the needle is indicatedby the needle gauge system. According to the Stubs Needle Gauge system,hypodermic needles in common medical use range from 7 gauge (thelargest) to 33 (the smallest). The word “fine,” as used herein, includesneedles ranging from 21 to 33 gauge (G), preferably 25 G to 31 G andmost preferably 25 G to 29 G. The definition for the fine hypodermicneedle applies to both re-usable and disposable types. Disposableneedles can be embedded in a plastic or aluminum hub that attaches tothe syringe barrel by means of a press-fit or twist-on fitting or the“Luer Lock” connections or be permanently attached to the syringebarrel.

As used herein, the term “heat-sensitive pharmacologically active agent”includes a pharmacologically active agent that can lose 3% or more ofits potency or concentration after autoclave treatment such as at 121°C. for 15-20 min. Some chemical drugs and many biological drugs areheat-sensitive. For these drugs, terminal sterilization procedures thatuse heat (or autoclaving) are not feasible.

As used herein, the term “injectable or extrudable” includes meeting theAcceptable Injectability Criterion as previously defined above.

As used herein, the term “insulin” includes a peptide hormone that iscentral to regulating carbohydrate and fat metabolism in the body,comprised of 51 amino acids and may be derived from various animalsources including bovine and porcine insulin or made by recombinanttechnology. The preferred insulin is a recombinant human insulin.

As used herein, the term “insulin analog” includes a chemically orenzymatically modified insulin wherein certain alteration is made to thepeptide sequence or amino acid side chains in order to alter thepharmacodynamic or pharmacokinetic property of the insulin. Thepreferred insulin analogs include insulin lispro, insulin aspart,insulin glulisine, insulin glargine, and insulin detemir The morepreferred insulin analog is insulin glargine or insulin detemir.

In accordance with the practice of the present invention, lecithins usedherein include pharmaceutical grade lecithins derived from egg orsoybean, which have been used in parenteral products and aresubstantially free from irritating, allergenic, inflammatory agents oragents that cause other deleterious biological reactions. Other examplesof phospholipids from naturally occurring sources that may be used forthis invention include sphingolipids in the form of sphingosine andderivatives (obtained from soybean, egg, brain & milk), gangliosides,and phytosphingosine and derivatives (obtained from yeast).

As used herein, the term “metal ion chelating agent or chelator”includes a metal ion chelator that is safe to use in an injectableproduct. A metal ion chelator works by binding to metal ions and therebyreduces the catalytic effect of metal ion on the oxidation, hydrolysisor other degradation reactions. Metal chelators that are useful in thisinvention may include disodium edetate (EDTA), glycine and citric acidand the respective salts thereof.

As used herein, the term “nanodispersion” includes an emulsion orsuspension formed by a homogenization step in the process for PG's. Ananodispersion of this invention contains phospholipid particles or oildroplets of a size less than 200 nm, preferably less than 100 nm andmost preferably less than 50 nm. A nanodispersion may be referred to asa “nanoemulsion” if oil is present or “nanosuspension” if oil is notpresent in the composition.

As used herein, the term “nanodispersion” includes a suspension oremulsion with with an average particle diameter of about 5 nm to about200 nm, preferably about 5 nm to about 100 nm and more preferably about5 nm to about 50 nm.

As used herein, the term “NPH insulin,” or NPH includes NeutralProtamine Hagedorn (also known as Humulin N, Novolin N, Novolin NPH, NPHLletin II, and insulin isophane) NPH is a suspension of crystalline zincinsulin combined with the positively charged polypeptide, protamine andwas created in 1936 when Nordisk formulated “isophane” insulin by addingNeutral Protamine to regular insulin. NPH insulin used herein alsoincludes other insoluble insulin particles formed with zinc and/orprotamine in ratios that are different from the insulin isophane.

As used herein, the term “oil” includes oil in a general sense toidentify hydrocarbon derivatives, carbohydrate derivatives, or similarorganic compounds that are liquid at body temperatures, e.g., about 37°C., and are pharmacologically acceptable in injectable formulations.“Oil” includes natural or synthetic glycerides or non-glyceridescomprising synthetic triglycerides such as tricaprylin, triolein, ortrimyristin, vegetable oil, animal oil, medium chain oil/glycerides,vitamin E, vitamin E acetate, vitamin E succinate, fatty acid, fattyacid monoester, cholesterol, and the like.

The term “one-phase” as used herein includes the ability of a PG tomaintain a substantially uniform content for its key component, i.e.,the pharmacologically active agent being uniformly distributedthroughout the gel matrix, even after centrifugation at 1000 RPM for 5minutes. In one aspect, a formulation that is one phase is“substantially uniform” wherein concentration of the pharmaceuticallyactive agent in the different samples collected throughout a gel in asyringe or a bulk has a coefficient of variation (CV) less than about1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.

The term “Phospholipid Gel” or “PG” as used herein includes a one-phase,transparent, translucent or opaque semi-solid mass (FIG. 1) thatcomprises 20-80% phospholipids and meets the “Acceptable InjectabilityCriterion.”

As used herein, the term “pH buffering agent” includes apharmaceutically acceptable pH buffer such as phosphate, acetate,citrate, bicarbonate, histidine, TRIS, and the like.

As used herein, the term “phospholipid” includes a lipid moleculecontaining one or more phosphate groups, including those derived fromeither glycerol (phosphoglycerides, glycerophospholipids) or sphingosine(sphingolipids). A phospholipid can be chemically synthesized orextracted from a natural source. Naturally occurring phospholipids aregenerally referred to as “lecithins ” According to the United StatePharmacopoeia (USP), lecithin is a non-proprietary name describing acomplex mixture of acetone-insoluble phospholipids, which are comprisedmainly of phosphotidylcholine, phosphotidylethanolamine,phosphotidylserine and phosphotidylinositol, combined with variousamounts of other substances such as triglycerides, fatty acids, andcarbohydrates.

As used herein, the term “primary dispersion” includes an emulsion orsuspension formed in the first step in a process of making the PG's ofthe present invention, that contain phospholipid particles or oildroplets of size greater than 500 nm in diameter. Such primarydispersions can be readily formed by simple mixing, such as stirring, orlow speed agitation. A dispersion may be referred to as an “emulsion” ifoil is present in the PG composition.

As used herein, the term “solubilizing agent” includes primarilycyclodextrins or surfactants such as polysorbate 80, bile salt and thelike.

As used herein, a “sugar” includes a safe and biocompatible carbohydrateagent that protects the nanodispersion during drying by maintaining thediscrete and sub-micron phospholipid particles. The sugars useful forthis invention include, but are not limited to, monosaccharides,disaccharides, polysaccharides, propylene glycols, polyethylene glycols,glycerols, poly-ols, dextrins, cyclodextrins, starches, celluloses andcellulose derivatives, or mixtures thereof For instance, in certainembodiments, the sugar is mannitol, sorbitol, xylitol, lactose,fructose, xylose, sucrose, trehalose, mannose, maltose, dextrose,dextran, or a mixture thereof In certain embodiments, the preferredsugar is sucrose.

As used herein, the term “tonicity/osmotic modifying agent” includes apharmaceutical additive that can be added to an injectablepharmacologically active agent and be used to adjust osmolality to closeto 300 mOsm. The tonicity/osmotic modifying agents useful in thisinvention include, but are not limited to, potassium or sodium chloride,trehalose, sucrose, sorbitol, glycerol, mannitol, polyethylene glycol,propylene glycol, albumin, amino acid and mixtures thereof.

II. Embodiments

The present invention provides a one-phase gel composition, comprising:

20 to 80% by weight of one or more phospholipids;

optionally a pharmacologically active agent; and

0.1 to 70% by weight water, wherein the gel composition is extrudablethrough a 25 G ½ inch long needle from a 1 cc syringe at an extrusionrate of 2 cc/min by an applied force of no more than 12 pounds. Thephospholipid depots are one-phase gels that can be aqueous orsubstantially anhydrous. Preferably, the invention is directed tocertain phospholipid compositions that are suitable for depotapplication.

Suitable synthetic phospholipids useful in the present inventioninclude, but are not limited to:

-   -   (1) Diacylglycerols, e.g. 1,2-Dilauroyl-sn-glycerol (DLG) and        Dimyristoyl-snglycerol (DMG);    -   (2) Phosphocholines, e.g.        1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and        1-Palmitoyl-2-01eoyl-sn-glycero-3-phosphocholine (POPC);    -   (3) Phosphoethanolamines, e.g.        1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) and        1,2-Palmitoyl-sn-glycero-3-phosphoethanolamine (POPE);    -   (4) Phosphoglycerols, e.g., Egg phosphatidylglycerol, sodium        salt (EPG, Na) and 1,2-Palmitoyl-sn-glycero-3-phospho glycerol,        sodium salt (POPG, Na);    -   (5) Phosphotidylserines, e.g.        1,2-Dimyristoyl-sn-glycero-3-phospho-L-serine, sodium salt        (DMPS,Na) and 1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine,        sodium salt (DPPS,Na);    -   (6) Mixed Chain Phospholipids, e.g.        1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and        1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol, sodium salt        (POPG,Na);    -   (7) Lysophospholipids, e.g.        1-Myristoyl-2-lyso-sn-glycero-3-phosphocholine (S-lyso-PC) and        1-Palmitoyl-2-lyso-sn-glycero-3-phosphocholine (P-lyso-PC); and    -   (8) Pegylated Phospholipids, e.g.        N-(Carbonyl-methoxypolyethyleneglycol 2000)-MPEG-2000-DPPE and        1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, sodium salt.

The preferred synthetic phospholipids are POPC and DMPC.

In accordance with the practice of the present invention, the selectionof a phospholipid for use in the depot compositions is determined byability of the phospholipid to (1) form a nanodispersion and maintainthe small particle size through the manufacturing process and afterwardsin storage, (2) be chemically compatible with the pharmacologicallyactive agent and (3) provide the desired depot or sustained releaseproperties for the pharmacologically active agent. Certain combinationsof phospholipids can be utilized to form the depot such as POPC andDMPC. An optional phospholipid or phospholipid combination for a depotcomposition can be selected using the physical and chemical screeningtest methods known to those skilled in the art.

In another embodiment, the PG compositions of the present inventioncomprise 2080% by weight, 25 to 70% by weight, and more preferably 30 to60% by weight of a phospholipid such as 25%, 30%, 35%, 40%, 45%, 50%,55%, or 60% by weight of a phospholipid or a mixture of phospholipids.

In one embodiment, a PG that contains a significant amount of water,i.e., about 10% to about 70%, such as 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, or 70%, and is referred to herein as “aqueousgel.”

In another embodiment, a PG is essentially or substantially free ofwater, i.e., such as less than 5%, preferably less than 3% and morepreferably less than 1%; such a PG is herein referred to as an“anhydrous gel.” The water content can be a de minimus amount or about0.01% to about 5%, or about 0.1% to about 5%, such as about 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%,3.5%, 4.0%, 4.5%, or about 5.0% w/w water.

In one embodiment, the present invention provides PG compositionscontaining pharmacologically active agents, satisfies the AcceptableInjectability Criterion and are able to deliver a pharmacologicallyactive agent in a prolonged and peak-less PK profile.

In one embodiment, the present invention provides PG compositionscompatible with heat-sensitive pharmacologically active agents ofbiological or synthetic chemical origin and methods of preparation ofsuch PG compositions that permit sterilization by filtration of thenanodispersion intermediate through a 0.2- or 0.45-micron pore membrane,thus eliminating the need for an aseptic process or terminalsterilization using heat or radiation.

In another embodiment, the present invention provides aqueous gel PGcompositions that are compatible with biological molecules such asinsulin or other biologically active proteins or peptides, and methodsto prepare such PG compositions, without the use of damaging amounts oforganic solvent. Biological molecules are easily denatured or destroyedby organic solvents. However, in accordance with the teachings of thepresent invention, a solvent-incompatible biological molecule can beformulated into aqueous gels as a sustained-release depot.

In another embodiment, the present invention provides PG compositionsthat are essentially devoid of water (i.e., the anhydrous gels) in orderto preserve water-sensitive pharmacologically active agents whileretaining the acceptable injectability properties in the final productand the desired 0.2- or 0.45-micron sterile filtration step in theprocess.

In a preferred embodiment, the aqueous gels of the present inventioncomprises a biological molecule such as a protein, a peptide, a nucleicacid sequence, a virus, a cell line or a hydrophilic chemical drug orsalt or solvate, and combinations thereof.

In another preferred embodiment, the PG gel of the present invention,either aqueous or anhydrous, comprises a heat-sensitivepharmacologically active agent such as a protein, a peptide, a nucleicacid sequence, a virus, a cell line or a sensitive chemical drug, whichwould be degraded or destroyed by heat or radiation typically used forterminal sterilization.

In yet another embodiment, the present invention provides an anhydrousgel that contains a water-sensitive pharmacologically active agent.

In yet another embodiment, the present invention provides anhydrous gelsthat contain lipophilic or water-insoluble pharmacologically activeagents.

The present invention provides an anhydrous gel that can be used todissolve highly water-soluble or hydrophilic pharmacologically activeagents despite the fact that the gel is essentially water-free. Forexample, certain pharmacologically active agents in their salt forms,such as a sodium salt (e.g., sodium heparin) or a hydrochloride salt(e.g., lidocaine HCl), are extremely water-soluble and have very lowsolubility in oil or lipid. The invention methods to prepare anhydrousgel disclosed herein have allowed surprisingly high solubilization ofsuch a highly hydrophilic pharmacologically active agents in anhydrousgels that essentially contain no water (EXAMPLES 5 and 6). The lidocaineHCl anhydrous gel is transparent and free of any insoluble solidparticles. In contrast, using conventional methods to mix lidocaine withthe other components of the anhydrous gel results in a suspension havingmost of the pharmacologically active agent remaining undissolved. Thisunexpected dissolution property, together with the absence of water ofthe anhydrous gel, provides an advantageous utility for forming depotcompositions that contain pharmacologically active agents that arewater-soluble yet sensitive to water, such as insulin and interferon.

The present invention provides aqueous gels that can be used to dissolveextremely water-insoluble or hydrophobic pharmacologically active agentsdespite the fact that the gels contain 20-70% water. For example,hydrophobic pharmacologically active agents such as docetaxel can bereadily dissolved in an aqueous gel (EXAMPLE 7) and the resulting gel istransparent and free of any insoluble solid particles. This is incontrast to conventional methods, which would form a suspension withmost of the hydrophobic pharmacologically active agent remainingundissolved following its addition into an aqueous composition. Thisunexpected dissolution property, together with the absence of solvent topromote the dissolution, provides an advantageous utility for formingdepot compositions containing water-insoluble pharmacologically activeagents without solvent or solvent-related safety concerns.

Table I below summarizes some, but not all, representative classes ofthe pharmacologically active agents that can be formulated as depots bythe present invention.

TABLE I Classes of pharmacologically active agents Water- soluble Stablein Heat- Exemplary pharmacologically Applicable (Hydrophilic) watersensitive active agents PG Yes Yes Yes Insulin (EXAMPLES 2-5, 19,Aqueous Gel 30, and 31) & Anhydrous Gel Yes No Yes Buprenorphine HC1(EXAMPLE Anhydrous 6) Gel No No Yes Docetaxel (EXAMPLE 7) Anhydrous GelYes Yes No Lidocaine (EXAMPLE 8) Anhydrous Gel Yes Yes Yes Exenatide(EXAMPLE 9) Aqueous Gel Yes No Yes Beta Interferon (EXAMPLE 10) AqueousGel Yes Yes Yes Heparin (EXAMPLE 11) Aqueous Gel Yes Yes Yes EpotinAlpha (EXAMPLE 12) Aqueous Gel Yes No Yes Human Growth Hormone Anhydrous(EXAMPLE 13) Gel Yes No Yes Adalimumab (EXAMPLE 14) Anhydrous Gel Yes NoYes Cefazolin & Metronidazole Anhydrous (EXAMPLE 15) Gel Yes No YesBupivacane (EXAMPLE 16) Anhydrous Gel No Yes No Predisone (EXAMPLE 20)Anhydrous Gel No Yes No Ibuprofen (EXAMPLE 21) Anhydrous Gel No Yes NoClotriamazole (EXAMPLE 22) Anhydrous Gel No Yes Yes Risperidone (EXAMPLE23) Anhydrous Gel No No No Tamoxifen citrate (EXAMPLE 24) Anhydrous GelNo Yes No Diazepan (EXAMPLE 25) Anhydrous Gel Yes Yes Yes Insulindetermir (EXAMPLE 30) Aqueous Gel No No Yes NPH Insulin (EXAMPLE 31)Aqueous Gel Yes Yes Yes BOTOX ® (EXAMPLE 32) Aqueous Gel

In a preferred embodiment, the phospholipid may be a lecithin, asynthetic phospholipid, or mixtures thereof. The preferred concentrationof phospholipid is 20 to 80%, preferably 25 to 60%, and more preferably30 to 50% such as 30%, 35%, 40%, 45%, or 50% of the PG weight.

In a preferred embodiment, oil may be used in the present invention's PGcompositions. The oil may be synthetic triglycerides such astricaprylin, trimyristin or triolein, vegetable oil, medium chain oil,vitamin E, vitamin E acetate, vitamin E succinate, oleic acid or otherunsaturated fatty acids or their monoetsers (e.g., ethyl oleate) orcholesterol, or mixtures thereof. The preferred oils are sesame oil,medium chain oil, ethyl oleate and the synthetic triglycerides and thepreferred concentration of oil is 1 to 50%, preferably 2 to 20% and morepreferably 5 to 10% of the PG weight, such as 5%, 6%, 7%, 8%, 9% or 10%.

In a preferred embodiment, a sugar can be used in the present PGcompositions. The sugar may be sucrose, dextrose, lactose, glucose,trehalose, maltose, mannitol, sorbitol, glycerol, amylose, starch,amylopectin or mixtures thereof. The preferred sugars are sucrose andglycerol.

The preferred concentration of sugar is 0.5 to 20%, preferably 1 to 15%and more preferably 2 to 10% such as 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or10% of the PG weight.

In a preferred embodiment, a solvent can be used in the invention PGcompositions. The solvent may be ethanol, propylene glycol, glycerol,sorbitol, polyethylene glycol, silicone oil, glycofurol, ethyl oleate,or mixtures thereof. The preferred solvents are ethanol, glycerol andpropylene glycol. The preferred concentration of solvent is 0.5 to 20%,preferably 1 to 15% and more preferably 2 to 10% such as 2%, 3%, 4%, 5%,6%, 7%, 8%, 9% or 10% of the PG weight.

In one embodiment, the invention PG compositions may contain water. Thepreferred concentration of water is about 10 to 70% of the aqueous geldepot weight, and less than about 5%, preferably less than 3% or mostpreferably less than 1% of the anhydrous gel depot weight.

In one embodiment, the invention PG compositions may comprise afunctional pharmaceutical excipient such as acidifying agents,alkalizing agents, pH buffering agents, metal ion chelators,antioxidants, stabilizers, preservatives, tonicity/osmotic pressuremodifiers, condensing agents, or a mixture thereof The selection of afunctional excipient(s) in a PG composition can be made based onstability requirement or other pharmaceutical considerations known bythose skilled in the art. Example excipients include, but are notlimited to, HCl or NaOH for the pH adjuster, acetate or histidine for pHbuffer, EDTA for the metal ion chelator, vitamin E, ascorbic acid orcysteine for the antioxidant, methionine for the stabilizer,meta-cresol, phenol or benzyl alcohol for the preservative, sodiumchloride, glycerol or sucrose for the tonicity/osmotic pressuremodifier, etc.

In one embodiment, the amount of active agent is about 0 to 20% byweight a pharmacologically active agent. In one embodiment, theinvention PG composition does not contain any pharmacologically activeagent or drug and such “drug-free” PG may be used as a tissue filler oras a wound salve. In this embodiment, the optional pharmaceutical activeingredient is absent or not present.

In other embodiments, the PG contains about 1.0×10⁻⁷% to about 1% byweight of a pharmacologically active agent. In other embodiments, theactive agent is about 1 to about 20% such as 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20% by weight of apharmacologically active agent. In other embodiments, the amount isabout 1% to about 6% or 3% to about 8% or even about 4% to about 11% byweight a pharmacologically active agent.

In one embodiment, the PG may contain about 0.1 nanogram/g up to 10nanogram/g, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,3, 4, 5, 6, 7, 8, 9, or 10 ng/mL of an active ingredient such as Botox®,or about 0.6 to 4.8 nanogram/g or even about 0.6 to 4.8 nanogram/g ofthe formulation. In certain instances, the amount of active ingredientis around 1.0×10⁻⁷% by weight. In certain instances, the active agent ispresent at about 1.0×10⁻⁷% to about 1% by weight.

Certain embodiments are preferred. For example, in one embodiment, thepresent invention provides a one-phase aqueous gel compositioncomprising:

-   -   20 to 80% by weight, preferably 25 to 70% by weight, and more        preferably 30 to 60% by weight one or more phospholipid(s);    -   10 to 70% by weight, preferably 20 to 60% by weight, and more        preferably 40 to 60% by weight of water; and    -   0 to 20% by weight a pharmacologically active agent, wherein the        gel composition requires an applied force of no more than 15        pounds to be extruded from a 1 cc syringe through a 25 G ½ inch        long needle at a rate of 2 cc/min.

In another embodiment, the present invention provides a one-phaseanhydrous gel comprising:

-   -   20 to 80% by weight, preferably 25 to 70% by weight, and more        preferably 30 to 60% by weight one or more phospholipid(s);    -   5 to 60% by weight, preferably 10 to 50% by weight, and more        preferably 20 to 40% by weight a non-aqueous component selected        from groups comprising sugar, oil, or solvent, and    -   0 to 20% by weight a pharmacologically active agent, wherein the        gel composition requires an applied force of no more than 15        pounds to be extruded from a 1 cc syringe through a 25 G ½ inch        long needle at a rate of 2 cc/min.

Further, in accordance with the present invention, there are providedone-phase gel compositions comprising:

-   -   (1) 20 to 80% by weight, preferably 25 to 50% by weight, and        more preferably 30 to 40% by weight one or more phospholipid(s),        and    -   (2) 100 to 700 IU/g an insulin, insulin analog, a crystalline        insulin with zinc and/or protamine, an NPH insulin or a        combination thereof and water, wherein the gel composition        requires an applied force of no more than 15 pounds to be        extruded from a 1 cc syringe through a 25 G Y2 inch long needle        at a rate of 2 cc/min and maintains plasma glucose concentration        below 130 mg/dl for no less than 18 hours following a        subcutaneous injection of 20 IU/kg insulin dose into        streptozotosin-induced type-I diabetic rats.

In a preferred embodiment, the PG compositions contain at least onepharmacologically active agent or drug. Suitable “pharmacologicallyactive agents” contemplated for use herein are not limited bytherapeutic category. Pharmacologically active agents can be smallmolecules made by synthetic chemistry or extraction (“chemical drugs”)or biological drugs including proteins, peptides, oligonucleotides,viruses, cells, and the like. The PG compositions of the presentinvention have particular utility for heat-sensitive pharmacologicallyactive agents, especially the biological drugs, such as insulin.

The preferred chemical drugs include, but are not limited to,antibiotics, anticancer agents, anesthetics, analgesics, hormones,antidiabetics and metabolic disorder drugs, with examples includingcefazolin, metronidazole, bupivacaine, lidocaine, buprenorphine,paclitaxel, and docetaxel. The term chemical drugs also include salts,solvates isomers, active metabolites, or combinations of the chemicaldrugs.

The biological drugs contemplated for this invention include, but arenot -limited to, biologically active agents selected from (a) bloodproteins such as factor IXa, hemoglobin, protein C; (b) antibioticpeptide such as bactericidal/permeability-increasing protein (Bpi),magainin, peptidyl mimetics, protegrin, ramoplanin; (c) enzymes such ascomasain, transforming growth factor, alpha-L-iduronidase,galactosidase, gelonin, glutamic acid, decarboxylase, ribonuclease, tpavariants; (d) antibodies such as anti-EFGr, anti-lymphoma antibody,anti-Her2, anti-Cd11/Cd18 integrin, anti-integrin receptors, anti-Cd52;(e) hormones such as amylin, extendin-4, relaxin, bone growth factors,epidermal growth factor, fibroblast growth factor, hematopoietin,insulin, insulin-like growth factor-I, leptin, natriuretic peptides,neural growth factors, parathyroid hormone, thrombopoietin, thymosinalpha-1: (f) enzyme inhibitors such as angiostatin and endostatin,bivalirudin, nematode anticoagulant proteins; (g) vaccines; (h)lymphokines such as interleukin-4, interleukin-6, interleukin-10,interleukin-12, (h) stem cell factor; (i) myeloid progenitor inhibitoryfactor-1, macrophage colony-stimulating factor, botulinin, fusionproteins, collagen, surfactant protein, protamine sulfate and heparin.The team biological drugs also include salts, solvates isomers, activemetabolites, or combinations of the biological drugs.

The preferred biological drugs include, but are not limited to, insulin,interferon, growth hormone, calcitonin, parathiroid hormone, exernatide,pramlintide, heparin, granulocyte colony-stimulating factor (G-CSF),epoetin, adalimumab, trastuzumab, and mixtures thereof.

In one embodiment, the present invention provides PG compositions, whichcontain insulin and satisfies the Acceptable Injectability Criterion andare able to maintain a plasma glucose concentration below 130 mg/dl forno less than 18 hours following a subcutaneous injection of a 20 IU/kginsulin dose into streptozotosin-induced type-I diabetic rats.

In another embodiment, the insulin contained in the PG compositions ofthe present invention is of animal origin or is a recombinant insulin, ahuman recombinant insulin, a insulin complex with zinc, protamine or acombination thereof, an insulin analog or a mixture thereof.

In yet another embodiment, the PG composition of the present inventionthat contains insulin comprises 50 to 1000 IU/g, preferably 100 to 500IU/g, more preferably 100 to 400 IU/g such as 100 IU/g, 200 IU/g, 300IU/g, 350 IU/g or 400 IU/g or most preferably 100 IU/g insulin, insulinanalog or NPH insulin. In certain aspects, 100 IU is about 3.8 mg ofrecombinant human insulin. In still another embodiment, the PGcomposition of the present invention that contains insulin comprisesabout 0.1% to about 5%, preferably about 0.3% to about 5% such as 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0 or 5.0% insulin.

In certain embodiments, the one-phase gel composition of the presentinvention maintains a peak-less blood insulin vs time profile instreptozotosin-induced type-I diabetic rats within 24 hours following asubcutaneous injection of 20 IU/kg insulin, and wherein the insulinconcentration ratio of the highest point to the lowest is no more than6:1, no more than 5:1, no more than 4:1, or even no more than 3:1.

In one embodiment, the present invention provides certain PGcompositions that contain 20 to 80% by weight of one or morephospholipids and surprisingly satisfies or requires even less injectionforce than the Acceptable Injectability Criterion. In some otherembodiments, the present invention provides certain PG that requiresgreater injection force than the Acceptable Injectability Criterion andsuch PG's may be delivered through a large needle into certain bodycavity such as a surgical wound.

In another preferred embodiment, this invention relates to PGcompositions, in their injectable, stable and sterilized form, thatprovide a unique drug release profile that is prolonged and peak-less.Such release profile is highly desirable for certain drugs with shorthalf-lives, such as insulin, and permit them to be maintained atconstant levels in the circulation for a prolonged time. In a preferredembodiment, the PG compositions and achieve a 24 hr duration PD andpeak-less 24 hr duration PK profiles for human insulin.

In another preferred embodiment, the PG composition of the presentinvention has at least one of the properties listed below, and issuitable as a replacement for NPH basal insulin therapy:

-   -   1. Incorporates authentic human insulin (as in NPH);    -   2. Administered by the same route as NPH, i.e., subcutaneous        injection;    -   3. Delivers a 24 hr peak-less PK profile;    -   4. Provides a 24 hr PD profile that is significantly longer than        that shown for NPH;    -   5. Has a stability/shelf-life comparable to the NPH drug;    -   6. Is injectable through a fine needle, i.e., 25 G or smaller;    -   7. Has significantly improved dose-uniformity (i.e.,        injection-to-injection variability of <3% relative standard        deviation);    -   8. Is ready to use and does not require mixing prior to        injection;    -   9. Is injectable by pen injector;

In another embodiment, the PG compositions and methods of preparationdisclosed herein are useful for both synthetic and biological drugs. ThePG compositions are especially useful for biological drugs havingphysical and chemical properties similar to insulin, i.e., highly watersoluble, solvent-incompatible, and sensitive to heat or radiation.

The present invention provides PG compositions and their methods ofpreparation which have the following unexpected features:

-   -   (1) High phospholipid content (i.e., 20-80%).    -   (2) Surprisingly good injectability.    -   (3) Filterable through 0.2-0.4 micron filters to enable a        sterilization-by-filtration in the manufacturing process, thus        permitting PGs to be used with heat-and radiation sensitive        drugs.    -   (4) Compatible with water-soluble or solvent-incompatible        synthetic and biological pharmacologic agents.    -   (5) Prolonged and peak-less delivery profile-capable for certain        drugs such as insulin.

III. Methods of Making

Surprisingly, the aqueous gels and anhydrous gels that are preparedaccording to the methods of preparation of the present invention areeasily injectable through fine needles even with their high phospholipidcontent (e.g., 20-80%). In some formats, the aqueous gels or anhydrousgels are transparent in appearance and silky smooth to the touch.Theologically, these gels are shear thinning and thixotropic, which aredesired properties for good extrudability/injectability through a fineneedle. In contrast, the same compositions, when prepared by other knownprior art methods, result in thick pastes that are very difficult orimpossible to inject through a fine hypodermic needle (FIG. 4).

In one embodiment, the present invention provides a method for preparinga one-phase gel composition, the method comprising:

-   -   a) forming a primary dispersion comprising one or more        phospholipid(s) and an excessive amount of water;    -   b) homogenizing the primary dispersion to form a nanodispersion        with an average particle size of about 30 nm to about 200 nm in        diameter;    -   c) optionally passing the nanodispersion through a 0.2- or        0.45-micron filter; and    -   d) removing the excessive water to obtain a one-phase gel        composition.

In certain embodiments, the one-phase gel is an aqueous gel. In otherembodiments, the one-phase gel is substantially an anhydrous gel. Incertain preferred embodiments the one-phase gel further comprises apharmacologically active agent. When a pharmacologically active agent ispresent, it may be is added before step “b” or it may be added afterstep “b”. In other embodiments, it may be added before as well as afterstep b. In other embodiments, the present invention provides-a one-phaseaqueous gel (e.g., aqueous or a substantially anhydrous) made by methodsherein.

In addition, with regard to step “c” of passing the nanodispersionthrough a 0.2- or 0.45-micron filter, if included, the filtration stepcan be performed either before or after “removing the excessive water”step in making an aqueous or an anhydrous gel. Thus, in certain aspects,step “c” is included in the method, or in certain aspects, step “c” isperformed after step “d”. In certain aspects, the filtration step can beeliminated and the PG is sterilized by heat or radiation or preparedaseptically.

In another embodiment, the present invention relates to unique methodsto prepare sterile PG compositions that are filterable through a 0.2- to0.45-micron pore membrane to permit sterilization of the PG preparationby filtration, yet have a 20 to 80% by weight phospholipid content andare able to meet or require less force than the Acceptable InjectabilityCriterion.

In another embodiment, the present invention provides unique methods toprepare PG compositions to contain water-soluble or solvent-incompatibledrugs such as insulin without any precipitation or degradation of theinsulin, yet having about 20 to 80% by weight phospholipid content andrequire less injection force than the Acceptable InjectabilityCriterion.

More surprisingly, the aqueous gels prepared according to the presentinvention exhibit superior uniformity and physical stability over acomposition containing the same components, but prepared by methodstaught in prior art. FIG. 5 illustrates the superior uniformity andphysical stability of an aqueous gel produced according to EXAMPLE 1(right, T-4) over a paste resulting from mixing the same components butnot using the methods of the present invention to prepare the aqueousgel (left). After centrifugation (13,000 rpm, 10 minutes), the pasteproduced by a prior art method separates into liquid and solid phaseswhereas the aqueous gel prepared in accordance with the presentinvention remains as a uniform, single-phase gel (Example 27). Suchcontent uniformity is key for accurate dosing as well as the physicalstability required for adequate product shelf life for pharmaceuticalproducts.

In a preferred embodiment, a high-shear, high-energy or high-pressurehomogenizer (such as the microfluidizers from MicrofluidicsInternational Corporation) is used to convert the primary dispersion toa nanodispersion by reducing the phospholipid particles in the primarydispersion from more than 500 nm to less than 200 nm, preferable lessthan 100 nm and most preferably less than 50 nm. The reduction ofphospholipid particles greatly reduces viscosity and increases theinjectability of the final PG's. For example, before high-pressurehomogenization, a primary dispersion composition containing about 20%phospholipid is a white, opaque, thick yogurt-like mass and is notinjectable through a 25 G needle.

After homogenization in a microfluidizer to reduce the lipid diameter toabout 50 nm, the resulting nanodispersion is a clear, transparent, thinand water-like liquid with a remarkably reduced viscosity. Afterremoving the excessive water, the final PG satisfies the AcceptableInjectability Criterion. The nanodispersion can also be filtered througha 0.2- or 0.45-micron filter membrane, allowing sterilization of the PGpreparations prior to parenteral administration. In contrast, the samephospholipid-containing composition without the homogenization treatmentis not filterable through the same membranes.

In a preferred aspect, the present invention provides a method forpreparing a one-phase aqueous gel composition, comprising:

-   -   a) mixing the components to form a primary dispersion comprising        one or more phospholipid(s) and excessive water;    -   b) homogenizing the primary dispersion to form a nanodispersion        with an average particle size of less than about 200 nm in        diameter;    -   c) passing the nanodispersion through a 0.2- or 0.45-micron        filter; and    -   d) removing the excessive water to obtain the aqueous gel.

In another embodiment, the present invention provides a method forpreparing a one-phase anhydrous gel comprising:

-   -   a) mixing the components to form a primary dispersion comprising        one or more phospholipid(s), and excessive water;    -   b) homogenizing the primary dispersion to form a nanodispersion        with an average particle size of less than about 200 nm in        diameter;    -   c) passing the nanodispersion through a 0.2- or 0.45-micron        filter; and    -   d) removing water to less than 5%, preferably less than 3% and        more preferably less than 1% by wt of the anhydrous gel.

Additionally, in accordance with the present invention, there areprovided one-phase anhydrous gel compositions comprising:

-   -   a) mixing the components to form a dispersion comprising one or        more phospholipid(s), excessive water;    -   b) removing water to less than 5%, preferably less than 3% and        more preferably less than 1% by wt of the anhydrous gel;    -   c) adding a solvent;    -   d) mixing to obtain an anhydrous gel, and    -   e) passing the gel though a 0.2- or 0.45-micron filter.

In one particular aspect of making an anhydrous gel, it is not requiredto homogenize the primary dispersion. This is especially advantageouswith a PG formulation without an active ingredient or with a non-heatsensitive or radiation sensitive active ingredient.

According to the present invention, a primary dispersion contains atleast about 70-80% water, which is more than needed in the final PG's.However, this amount of water gives the dispersion the desired flowproperties in order to be processed in the microfluidizer. Once thenanodispersion is obtained, the excessive water is removed in order toachieve the final water content in the PG of 20 to 70% for an aqueousgel or less than about 5%, preferably about 3%, or more preferably about1% water content for an anhydrous gel so that the PG will have thedesired properties. In accordance with the practice of the presentinvention, it is important to maintain the small phospholipid particlesize during the water-removing (drying) step to maintain the lowviscosity or high injectability of the final PGs.

Emulsions or suspensions of phospholipids are thermodynamically unstablesystems. If not processed properly, the phospholipid droplets orparticles will aggregate, merge, grow in size and eventually result inthe phospholipid and separating the water phases (i.e., creaming out).When this happens the benefit of the reduced viscosity provided by thenanodispersion is lost. Surprisingly, in accordance with the practice ofthe present invention, the addition of certain sugars provides anunexpected protective effect for the nanodispersion against theaggregation of phospholipid particles or droplets during the waterremoval processes. The presence of sugar in the nanodispersion thuskeeps the phospholipid nanodispersion particle size essentiallyunchanged during the water removal step using the conditions disclosedherein.

In certain aspects, as shown in Examples 1 and 2, the resulting aqueousgels have about the same particle size as in the nanodispersion, whilemaintaining excellent injectability properties for injections throughfine hypodermic needles (FIG. 4). The present inventors have observedthat as water is removed from the nanodispersion to where the PG reachesa water content of 50% or less, a phase transition occurs that turns thesolution-like nanodispersion into a gel. The PG thus formed istransparent or translucent, one-phase and remains one-phase even afterbeing subjected to a strong separation force such as centrifugation.Upon mixing in water, the PGs of this invention can re-form thenanodispersion, suggesting that the PGs comprise discretenanometer-sized phospholipid particles.

In contrast, following other known prior art methods that simply mix thesame PG components, even with vigorous agitation for 24 hours, the samecompositions as in Example 1 and 2 produced a pasty mass, which isopaque, not one-phase and did not satisfy the Acceptable InjectabilityCriterion (FIG. 4).

Not wishing to be bound by a theory or mechanism of the invention, itappears that the superior injectability offered by the PG's of thisinvention is attributable to the extremely small phospholipid particlescreated by homogenization. This inventor speculates that by removing thewater from the nanodispersion, the nanometer sized phospholipidparticles stack together to form a certain organized structure like manysmall deformable “balloons” filled with oil and stacked together withwater in the interstitial space. As the water is removed, theinterstitial space is minimized causing the balloons to deform tocompress into each other to form a more rigid structure i.e., a gel, butrather than fusing into each other, the balloons remain discrete in thegel phase. When an external force is applied (such as from a syringeplunger), the gel easily deforms and conforms to the needle bore becauseof the very small and discrete phospholipid particles, thus allowing fora superior injectability. FIG. 9 is a schematic representation of thespeculated convention from a nanodispersion (left) to a PG (right) uponremoval of water. The dark dots depict the nanosized phospholipidparticles in the nanodispersion, and the space between the dots arefilled with water as in an aqueous gel or sugar or oil as in ananhydrous gel. As the water or solvent is removed, the particles becomestructurally organized into the gel.

Depending upon stability of the pharmacologically active agent and drugdelivery/release requirements, a pharmacologically active agent can beintroduced at a different step during the present invention's processaccording to the present methods.

In one embodiment, the present invention provides a method fordissolving the pharmacologically active agent in the aqueous phase thatcan then be mixed with the phospholipid to form the primary dispersionthat is subsequently carried through the rest of the process. Themethods may be used for a water-soluble pharmacologically active agentand when the pharmacologically active agent is shear stress-resistantand/or a slower drug release is desired.

In another embodiment, the present invention provides methods todissolve the pharmacologically active agent in an oil phase, whichcontains the phospholipids and, optionally oil, which can then be mixedwith the aqueous phase to form the primary dispersion that issubsequently carried through the rest of the process. This method may beused for a lipophilic, water-insoluble or fat-soluble pharmacologicallyactive agent.

In yet another embodiment, the present invention provides a method forintroducing the pharmacologically active agent into the primarydispersion prior to the homogenization step which is subsequentlycarried through the rest of the process.

In another embodiment, the present invention provides a method forintroducing the pharmacologically active agent into the nanodispersionafter the homogenization step which is subsequently carried through therest of the process.

In yet another embodiment, the present invention provides a method forintroducing the pharmacologically active agent into the gel after thewater removal step.

In one embodiment, the primary dispersion is made by mixing the oilphase containing phospholipid and other fat-soluble components with anaqueous phase which contains all water-soluble components.Alternatively, the primary dispersion is made by mixing all componentswith no particular order of addition.

In another embodiment, the oil phase is made by mixing the phospholipidand, optionally, oil and the pharmacologically active agent.Alternatively, the oil phase is made by dissolving the phospholipid, thepharmacologically active agent and, optionally, oil in a volatilesolvent such as ethanol and then removing the ethanol.

In one embodiment, the aqueous phase is made by mixing water, pHadjuster, pH buffer, chelator, antioxidant, stabilizer, preservative,and/or tonicity/osmotic pressure modifier to form a solution.Optionally, a pharmacologically active agent may be dissolved or addedto the aqueous phase.

In another embodiment, the filtration of the nanodispersion may beperformed using a vacuum filtration method, centrifugation filtration,or pressurized filtration method. Various models or makes of 0.2- or0.45-micron pore filter membranes are available. Examples includeSartopore, Sartobran P, Millipore, and the like. In some cases, apre-filter with a larger pore size may be used. The primary reason forthe filtration step is to sterilize the preparation.

In yet another embodiment, removal of water from the dispersion ornanodispersion can be done by various drying methods, for example, byrotational vacuum drying method or by sweeping the nanodispersion withair or nitrogen gas (“air drying”). The rotational vacuum drying can beperformed using commercially-available rotational evaporators such as aRotavap (Buchi). The air drying is accomplished by mechanically stirringthe nanodispersion while sweeping its surface with a stream of air ornitrogen gas. The air or nitrogen gas may be filtered through a 0.2- or0.45-micron pore filter to sterilize. Nitrogen gas is preferred if anycomponents in the composition are prone to oxidation.

In one embodiment, a solvent is added to faun the anhydrous gel, to 0.5to 20%, preferably 1 to 15% and more preferably 2 to 10% of theanhydrous gel weight to improve the injectability/filterability. Anexample of the suitable solvents is ethanol, which could be added from 1to 10% by weight. Prior to adding to the gel, the solvent may be firststerilized (e.g., by filtration through a 0.2- or 0.45-micron ratedfilter). After addition, the mixture is agitated to form a uniformanhydrous gel.

In another embodiment, the anhydrous gel containing the solvent issterilized by filtration through a 0.2- or 0.45-micron pore filter atthe end of the process.

In some embodiments, the aqueous or anhydrous gels are filled intosyringes to certain volume under aseptic conditions and are ready forinjecting after attaching needles to the syringes. The pre-filledsyringe format is convenient for self-administration. The preferredsyringe size is 1-10 mL and the preferred needle size is 25-29 G.

In one embodiment, the aqueous or anhydrous gels, after being injectedinto a soft tissue (e.g., subcutaneous or intramuscular injection),provide a slow drug release in vivo as shown by a prolonged plasma drugconcentration-time profile, compared to the same pharmacologicallyactive agent without a depot composition. The preferred profile covers1, 3, 5, 7, 10 and 14 days (FIG. 2, FIG. 7 & FIGS. 8A-8B).

In another embodiment, the aqueous or anhydrous gels, after beinginjected into a soft tissue (e.g., subcutaneous or intramuscularinjection), provide a prolonged drug residence time at the injectionsite as shown by a maintained drug concentration or a sustained localdrug activity at the injection site-time profile compared to the samedrug without a depot composition. The preferred profile covers 1, 3, 5,7, 10 and 14 days (FIG. 3, FIG. 7 & FIGS. 8A-8B).

In certain aspects, the formulation has a dynamic viscosity of about100, 200, 500, 1000, 3000, and 5000 centipoise (cP). In certain aspects,the dynamic viscosity of the formulation is at about 5000, 10,000,50,000, 75,000, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸ or 1×10⁹ cP at STP. In yetother aspects, the formulation is thixotropic.

In certain aspects, the PG formulation of the present invention isacidic to neutral. In certain aspects, the formulation has a pH of about3 about 8.5. In certain other aspects, the formulation has a pH fromabout 5 to about 8.5, from about 5.5 to about 8.1, about 6 to about 7.9,about 5.5 to about 7.9, or from about 6.5 to about 7.5.

In a preferred aspect, the PG formulations of the present inventioncomprise a pH-adjusting agent. In one embodiment, the pH-adjusting agentis a base. Suitable pH-adjusting bases include amines (e.g.,diethanolamine or triethanolamine), bicarbonates, carbonates, andhydroxides such as alkali or alkaline earth metal hydroxides as well astransition metal hydroxides. The pH-adjusting agent is preferably sodiumhydroxide and is present in an amount sufficient to adjust the pH of thecomposition to between about pH 4.0 to about 8.5; more preferably, tobetween about pH 5.5 to about 7.0, such as 6.0 or 6.5. Alternatively,the pH-adjusting agent can also be an acid, an acid salt, or mixturesthereof In a preferred embodiment, the pH-adjusting agent is an acid.

Further, the pH-adjusting agent can also be a buffer. Suitable buffersinclude citrate/citric acid buffers, acetate/acetic acid buffers,phosphate/phosphoric acid buffers, formate/formic acid buffers,propionate/propionic acid buffers, lactate/lactic acid buffers,carbonate/carbonic acid buffers, ammonium/ammonia buffers, and the like.In certain embodiments, the buffer is an acidic buffer system (e.g.,benzocaine).

The invention will now be described in greater detail by reference tothe following non-limiting examples.

Example 1 Preparation of Aqueous Gels (Having No Pharmaceutically ActiveAgent) Using Lecithin

Components F-1 T-3 T-4 T-5 Compositions (Wt % in primary dispersion) (Wt% in PG) Sesame oil 4.0 6.6 8.0 10.1 Soy lecithin 15.0 24.7 30.2 38.0Cholesterol 0.6 1.0 1.2 1.5 Vitamin E 0.3 0.5 0.6 0.8 succinate (VES)Sucrose 5.0 8.2 10.1 12.7 De-ionized water 75.1 59.0 50.0 37.0(DI-water) Total 100 100.0 100.0 100.0 Name Supplier Grade Sesame oilCroda Super-refined, USP Soy lecithin LIPOID EP (Phospholipon 90G)Cholesterol Solvay Pharmaceuticals HP, NF VES Spectrum Chem. USP SucroseSpectrum Chem. NF De-ionized water Latitude Pharmaceuticals Inc.

Procedure

The F-1 primary dispersion first was prepared and converted to threeaqueous gels (T-3, T-4 and T-5) by the following procedure:

-   -   1. Weigh out sesame oil, Phospholipon 90 G and cholesterol into        a glass flask.    -   2. Add 50 mL ethanol USP.    -   3. Rotate the flask to dissolve all solids.    -   4. Vacuum dry to remove ethanol to less than 1% by weight.    -   5. Add VES (in a 5% stock solution), sucrose and DI-water to 500        g total weight.    -   6. Rotate the flask to mix to form a primary dispersion.    -   7. Homogenize the primary dispersion using a Microfluidics        International Corp Model M-110EH to obtain a nanodispersion.        Continue the process until the average particle diameter is        about 69 nm as determined by laser light scattering (Malvern        Nano Zetasizer). Record pH which is 5.9    -   8. Filter the nanodispersion through a 0.45-micron disposable        vacuum filter (Nalgene) in a biosafety hood to sterilize.    -   9. Aseptically, remove water by a rotational evaporator (Buchi        Model R-205 Rotavap) until the water content is about 59%, 50%        and 37% by weight to obtain respectively, T3, F-4 and T-5.

The T-3, T-4 and T5 were one-phase, uniform, and translucent/transparentgels (FIGS. 1A-1C) that were readily injectable and met the AcceptableInjectability Criterion defined herein. Their water contents wereconfirmed by a moisture balance. The average particle sizes after beingre-dispersed in water were determined as 63, 62 and 56 nm, respectively,for T-3, T-4 and T-5, as determined by laser light scattering. All threegels were filterable through 0.2-micron filters.

The compositions of this example can be used as tissue fillers forvarious indications such as cosmetic wrinkle removal.

Example 2 Preparation of Aqueous Gels Containing Recombinant HumanInsulin for Basal Insulin Therapy

Aqueous gels containing recombinant human insulin and 40%, 50% and 60%water were prepared using lecithin. Insulin was introduced into theprocess before microfluidization and the resulting nanodispersion wasfiltered for sterilization. Thus, no heat or radiation sterilization wasneeded in the process.

Compositions (% wt) Nanodispersio Aqueous Gel Component F-1 F-2 F-3 F-4Recombinant 74.9 (IU/g) 84.9 (IU/g) 100 (IU/g) 115.4 (IU/g) humaninsulin Sesame oil 4.0 6.43 8.03 9.64 Soy lecithin 15.0 24.10 30.1236.14 Cholesterol 0.6 0.96 1.20 1.45 Vitamin E 0.3 0.48 0.60 0.72succinate (VES) Sucrose 5.0 8.03 10.04 12.05 EDTA 0.015 0.018 0.0200.023 disodium dehydrate De-ionized 75.1 60 50 40 water (DI-water)

Procedure

The F-1 nanodispersion was first prepared and converted to threeanhydrous gels (F-2, F-3 and F-4) containing 40, 50 and 60% water asfollows:

-   -   1. Weigh out sesame oil, soy lecithin and cholesterol into a        glass flask.    -   2. Add ethanol USP.    -   3. Rotate the flask to dissolve all solids.    -   4. Vacuum dry to remove ethanol to less than 1% by weight.    -   5. Add VES (in a 5% stock solution), sucrose, EDTA and DI-water.    -   6. Add a recombinant human insulin stock solution (Humulin R        U100 by Eli Lilly and Co.).    -   7. Mix to form a primary dispersion.    -   8. Adjust pH to 6.8 using NaOH/HCl.    -   9. Homogenize the primary dispersion using a Microfluidics        International Corp. Model M-110EH to obtain a nanodispersion.        Continue the process until the average particle diameter is        about 88 nm as determined by laser light scattering (Malvern        Nano Zetasizer).    -   10. Filter the nanodispersion through a 0.2-micron filter        (Millipore Sterflip) in a biosafety hood to sterilize the        nanodispersion.    -   11. Aseptically, remove water using a rotational evaporator to        reach water content at 60% to obtain the F-2 gel. Continue the        drying process to 50% water for F-3, and 40% for F-4.

The F-2, F-3 and F-4 aqueous gels were one-phase and translucent gels.All satisfied the Acceptable Injectability Criterion. The insulinconcentration and integrity were confirmed by an RP-HPLC analysisaccording to USP.

Example 3 Preparation of Aqueous Gels Containing Recombinant HumanInsulin for Basal Insulin Therapy

The procedure was directed to preparation of aqueous gels containingrecombinant human insulin at 100 IU/mL and 50% water using a soylecithin (F-1G) and a synthetic phospholipid (F-5G). The insulin wasintroduced into the process before microfluidization.

Component F-1G F-5G Compositions (% wt) (Nanodispersion) (Gel)(Nanodispersion) (Gel) Recombinant human insulin 0.284 0.379* 0.2840.379* Sesame oil 4 8.03 4 8.03 Phospholipon 90G (PL90G) 15 30.121-Palmitoy1-2-01eoyl-sn-glycero-3- 15 30.12 phosphocholine (POPC)Cholesterol 0.6 1.2 0.6 1.2 Vitamin E succinate (VES) 0.3 0.6 0.3 0.6Sucrose 5 10.04 5 10.04 EDTA disodium dehydrate 0.011 0.015 0.011 0.015Histidine 0.078 0.104 0.078 0.104 De-ionized water (DI-water) 75.1 5075.1 50 *Equivalent to 100 IU/g

Procedure

The nanodispersions were first prepared and converted to the anhydrousgels following the steps below:

-   -   1. Weigh out sesame oil, PL9OG or POPC and cholesterol into a        glass flask.    -   2. Add ethanol USP.    -   3. Rotate the flask to dissolve all solids.    -   4. Vacuum dry to remove ethanol to less than 1% by weight.    -   5. Add VES (in a 5% stock solution), sucrose, EDTA, histidine        and DI-water.    -   6. Add recombinant human insulin powder (Incelligent SG by        Millipore, 26.4 USP unit/mg)    -   7. Mix to form a primary dispersion.    -   8. Adjust pH to 7 using NaOH/HCl.    -   9. Homogenize the primary dispersion using a Microfluidizer        Model M-110EH to obtain a nanoemulsion. Continue the process        until the average particle diameter is about 53 nm as determined        by laser light scattering (Malvern Nano Zetasizer).    -   10. Filter the nanodispersion through a 0.2-micron filter in a        biosafety hood for sterilization.    -   11. Aseptically, remove water by a rotational evaporator until        the water content is about 50% to obtain an F-1G or F-5G aqueous        gel.

The F-1G and F-5G were one-phase, colorless (F-5G) and transparent gels.Both met the Acceptable Injectability Criterion (FIGS. 1A-1C). Watercontents were confirmed by thermogravimetric analysis.

Example 4 Preparation of Recombinant Human Insulin Aqueous GelsContaining Additional Functional Excipients for Basal Insulin Therapy

The following compositions were made using the same procedure asdescribed in EXAMPLES 1 to 3 to prepare aqueous gels, but containedrecombinant human insulin at 100 IU/mL and water at 50% water. However,a synthetic phospholipid and various other excipients were added in theaqueous phase to increase stability. Sucrose, EDTA disodium dehydrate,M-cresol, phenol, L-histidine, L-cysteine, zinc (as zinc chloride),and/or protamine sulfate were dissolved in the aqueous phase first. Theinsulin was introduced into the process before the microfluidizationstep.

Compositions % Wt F-6G F-7G F-8G F-9G Recombinant Human Insulin 0.3790.379 0.379 0.379 powder (at 26.4 U/mg) Sesame oil 8 8 8 8 POPC 30 30 3030 Cholesterol 1.2 1.2 1.2 1.2 VES 0.6 0.6 0.6 0.6 Sucrose 10 10 10 10EDTA disodium dehydrate 0.1 M-CRESOL 0.16 0.16 0.16 0.16 Phenol 0.0650.065 0.065 0.065 L-Histidine 0.1 0.1 0.1 0.1 L-cysteine 0.1 0.1 Zinc0.0025 Protamine sulfate 0.024 Water DI- 49.40 49.50 49.40 49.37 Total100 100 100 100

F-6 G, F-7 G, F-8 G and F-9 G were one-phase, colorless andtransparent/translucent (or opaque in the case of F-9G) gels. All werereadily injectable through a 26 G needle. The insulin concentration andintegrity in each gel were confirmed by an RP-HPLC analysis according tothe standard USP method.

Example 5 Preparation of Anhydrous Gels Containing Humulin R and HumulinNPH for Basal Insulin Therapy

The procedure was directed to preparation of anhydrous gels containingrecombinant human insulin (Humulin R) and recombinant human insulinzinc/protamine complex (Humulin N, or NPH) at about 100 IU/g. Humulin Ror Humulin NPH are insoluble in oil, incompatible with organic solventsand are heat-sensitive. This method allows dissolution or incorporationof these hydrophilic pharmacologically active agents into a sterilizedwater-free anhydrous gel without using a terminal heat sterilizationstep in the process.

Composition (% wt) Component S-4 S-9 Humulin R 100 IU/mL / Humulin NPH /100 IU/mL Soy lecithin 54 54 Sesame oil 40 40 Ethanol  6  6

Procedure

A 1 g batch for S-4 or S-9 was prepared as follows:

-   -   1. Weigh out sesame oil, soy lecithin and water-for-injection        into a plastic tube.    -   2. Homogenize using a Beadbeater to form a nanodispersion.    -   3. Pass through a 0.2 micron pore filter.    -   4. Add Humulin R solution or Humulin NPH suspension to the        filtered emulsion. Mix well.    -   5. Lyophilize the nanodispersion to remove water to less than        1%.    -   6. Add ethanol.    -   7. Mix well to obtain an Anhydrous Gel (S-4 and S-9).

The S-4 was a one-phase and translucent gel and S-9 was a one-phaseopaque gel. Both gels were readily injectable through a 26 G needle. Theinsulin strengths in these gels were confirmed by HPLC analysis.

Example 6 Preparation a Long-acting Depot Comprising an Anhydrous GelContaining Buprenorphine Hydrochloride

The procedure was directed to preparation of an anhydrous gel containingwater-soluble anesthetic buprenorphine hydrochloride. Buprenorphinehydrochloride is insoluble in oil and is heat-sensitive. This methodallows a complete dissolution/incorporation of this hydrophilicpharmacologically active agent in a water-free anhydrous gel without theneed for a terminal heat sterilization step in the process.

Composition (% wt) Component F-27 Buprenorphine HC1 0.521,2-Dimyristoyl-sn-glycero-3-phosphoglycerol, 0.71 ammonium/sodium salt(DMPG) Phospholip on 90G 55.49 Castor oil 36.99 Benzyl alcohol 1.00Ethanol 5.00 EDTA disodium dehydrate 0.05 Sodium phosphate monobasic0.24

Procedure

-   -   A 10 g (final gel weight) batch of F-27 was prepared as follows:    -   1. Weigh out castor oil, lecithin, benzyl alcohol, bupronorphine        HCl and DMPG into a plastic bottle. Mix to form an oil phase.    -   2. Weigh out EDTA, sodium phosphate monobasic and        Water-for-Injection, USP (WFI) in a separate container; shake to        dissolve all solids and adjust pH to 7 to obtain an aqueous        phase. Filter the aqueous phase.    -   3. Add the aqueous phase to the oil phase. Shake vigorously to        form a primary dispersion.    -   4. Homogenize by sonication to form a nanodispersion.    -   5. Freeze-dry the nanodispersion to remove water to less than        2%.    -   6. Add ethanol.    -   7. Mix well to obtain an Anhydrous Gel (F-27).

F-27 was a one-phase, opaque gel that was readily injectable through a26 G needle. The buprenorphine strength in this gel was confirmed byHPLC and the water content confirmed using thermogravimetric analysis.The resultant gel is intended as a long-acting depot (e.g., once-a-weekdosing) for systemic analgesia or for treatment of narcotic drug abuse.

Example 7 Preparation of an Aqueous Gel Containing Docetaxel

The procedure was directed to preparation of an aqueous gel (F-177)containing a highly water-insoluble drug docetaxel and 50% water using asoy lecithin. Docetaxel was introduced into the process beforemicrofluidization. This docetaxel gel depot is for intratumor injectionto provide a prolonged anticancer activity.

Compositions (% wt) F-177 Primary Component dispersion Gel Docetaxeltrihydrate 0.600 0.811 Miglyol 812 4.000 5.405 Soybean oil 4.000 5.405Soy lecithin 10.000 13.514 Cholesterol 0.6 0.811 Vitamin E succinate(VES) 0.3 0.404 Sucrose 17.5 23.649 Water-for-Injection 67 50 1NNaOH/HC1 adjust pH to 7.6

Procedure

A 500 g (primary dispersion wt) batch was prepared and converted to anaqueous gel as follows:

-   -   2. Weigh out docetaxol trihydrate, Miglyol 812, soybean oil, soy        lecithin and cholesterol into a glass flask.    -   3. Add 500 mL ethanol USP 200 proof.    -   4. Rotate the flask to dissolve all solids at 50° C.    -   5. Vacuum dry to remove ethanol to less than 2% by weight.    -   6. Add VES (in a 5% stock solution), sucrose and DI-water.    -   7. Mix to form a primary dispersion.    -   8. Adjust pH to 7.6+/−0.2 using NaOH/HCl.    -   9. Homogenize the primary dispersion using a Microfluidics        International Corp. Model M-110EH to obtain a nanodispersion.        Continue the process until the average particle diameter is        about 100 nm as determined by laser light scattering (Malvern        Nano Zetasizer).    -   10. Filter the nanodispersion through a 0.2-micron pore filter        in a biosafety hood to sterilize the nanodispersion.    -   11. Aseptically, remove water from the nanodispersion using a        rotational evaporator until the water content is about 50% to        obtain the F-177 aqueous gel.

Example 8 Preparation of a Long-acting Depot Comprising an Anhydrous GelContaining Lidocaine

This procedure was directed to preparation of an anhydrous gelcontaining the highly water-soluble local anesthetic lidocaine. Thismethod also allows incorporation of the water-soluble (andfat-insoluble) excipients (i.e., EDTA disodium dihydrate and potassiummonobasic phosphate, KOH) in the anhydrous gel.

Composition (% wt) Component F-20 Lidocaine 2 Soy lecithin 45 Sesame oil44 EDTA disodium dehydrate 0.10 Potassium monobasic phosphate 0.14Benzyl alcohol 1 Ethanol 4 1N KOH pH to 7.0

Procedure

A 20 g (final gel weight) batch was prepared as follows:

-   -   1. Weigh out lidocaine, sesame oil, soy lecithin and benzyl        alcohol into a glass flask.    -   2. Add ethanol USP 200 proof and rotate the flask to dissolve        all.    -   3. Vacuum dry to remove ethanol to less than 1% by weight.    -   4. Add KH₂PO₄, EDTA and DI-water.    -   5. Homogenize to form a nanodispersion.    -   6. Adjust pH to 7.0+/−0.2 using NaOH/HCl.    -   7. Sterile filter the nanodispersion through a 0.2 micrometer        pore filter.    -   8. Lyophilize the nanodispersion to remove water to less than        2%.    -   9. Add ethanol.    -   10. Mix well to obtain an anhydrous Gel (F-20).

The F-20 is a one-phase, slightly yellow and translucent gel that isreadily injectable through a 26 G needle.

Example 9 Preparation of an Aqueous Gel Containing Exenatide as aLong-Acting Depot

An aqueous gel depot is prepared to contain about 2 mg/mL exenatideusing a composition and method as described in EXAMPLE 3. The resultantgel is a long-acting depot (e.g. once-a-week dosing) of thisanti-diabetic drug.

Example 10 Preparation of a Long-acting Depot Comprising an Aqueous GelContaining Interferon Beta-1 a.

An aqueous gel depot is prepared to contain about 35-350 microgram/mLinterferon beta-1a using a composition and method as described inEXAMPLE 3 and 4. The resultant gel is a long-acting depot (e.g.,once-a-week dosing) and reduced injection site reaction of thisanti-viral, antiproliferative and immunomodulatory drug.

Example 11 Preparation of a Long-acting Depot Comprising an Aqueous GelContaining Heparin

An aqueous gel is prepared to contain about 40-500 microgram/mLenoxaparin sodium (Lovenox) using a composition and method as describedin EXAMPLES 3 and 4. The resultant gel is a long-acting depot (e.g.once-a-week dosing) for prophylaxis and treatment of deep veinthrombosis (DVT), ischemic angina and myocardial infarction.

Example 12 Preparation of Long-Acting Depot Comprising an Aqueous GelContaining Epoetin alpha

An aqueous gel depot is prepared to contain about 10-30K units/mLEpoetin alpha (EPOGEN®) using a composition and method as described inEXAMPLES 3 and 4. The resultant gel is a long-acting depot (e.g.,once-a-week dosing) for erythrocytopenia.

Example 13 Preparation of Long-Acting Depot Comprising an Anhydrous GelContaining Growth Hormone

An anhydrous gel is prepared to contain about 5-50 mg/mL human growthhormone (somatropin) using a composition and method as described inEXAMPLES 5 and 6. The resultant gel is a long-acting depot (e.g.once-a-week dosing) for growth hormone deficiency in pediatric and adultpatients, Turner syndrome and SHOX deficiency.

Example 14 Preparation of Long-Acting Depot Comprising an Anhydrous GelContaining Adalimumab

An anhydrous gel depot is prepared to contain 1 to 20% adalimumab(Humira) using a composition and method as described in EXAMPLES 5 and6. The resultant gel is a long-acting depot (e.g. once-a-week dosing)for treatment of arthritis, psoriasis, ankylosing spondylitis andCrohn's disease.

Example 15 Preparation of a Long-Acting Depot Comprising an AnhydrousGel Containing Cefazolin, Metronidazole or a Combination Thereof

An anhydrous gel depot is prepared to contain about 5 to 20% Cefazolin,50 mg/mL Metronidazole or a combination thereof using a composition andmethod as described in EXAMPLES 5 and 6. The resultant gel is along-acting depot following instillation into surgical wound orinjection into the soft tissue near the surgical wound to prevent postsurgical infection.

Example 16 Preparation of a Long-acting Depot Comprising an AnhydrousGel Containing Bupivacaine

An anhydrous gel depot is prepared to contain about 40 mg/mL bupivacaineHCl using a composition and method as described in EXAMPLES 5 and 6. Theresultant gel is a long-acting depot following instillation into asurgical wound or injection into the soft tissue surrounding thesurgical wound to alleviate post surgical pain.

Example 17 Prolonged and Peak-less Pharmacokinetic Profile by anAnhydrous Gel Containing Buprenorphine HCl in Dogs

A pharmacokinetic study was conducted where 6 dogs were administered at0.25 mg/kg dose by subcutaneous or intramuscular injection of the F-27anhydrous gel depot as described in EXAMPLE 6. Plasma samples were takenand the buprenorphine content analyzed by LC-MS. The results are shownin FIG. 2. The plasma profiles reveal that F-27 depot provided aprolonged in vivo release for at least 5 days with a low initial burst.The prolonged, controlled and essentially peak-less PK profile can be avery desirable feature for drugs where a high C_(max) may cause adverseeffects.

Example 18 Prolonged Local Analgesic/Anesthetic Efficacy of Lidocainefrom an Anhydrous Gel Prepared According to the Method in EXAMPLE 8Following Intracutaneous Injection in Guinea Pigs

This study was intended to compare the prolonged localanalgesic/anesthetic activity of lidocaine in the F-20 depot formulationto an immediate release solution formulation in guinea pig pinprick painmodel. An anhydrous gel (F-20 composition) containing the highly watersoluble (and oil insoluble) lidocaine was prepared as described inEXAMPLE 8 and a solution formulation (“Control”) was also prepared basedon the Lidocaine Solution for Injection, USP. Male guinea pigs between300˜350 grams in body weight were used. The anesthesia/analgesiaactivity was determined using the intracutaneous wheal pinprick model asdescribed in U.S. Pat. No. 6,045,824 by Kim. On the day preceding theinjection, the backs of the animals were clipped. Each animal received a0.25 mL intracutaneous injection of the lidocaine formulation. Thereaction to pinpricks at the site of injection was tested just prior toinjection (pre-injection) and at specific time points after theinjection. The pinpricks were to be applied first to an area outside thewheal at each time point for positive control. After observing theanimals' normal reaction to the pinprick (vocalization response), sixpricks were applied inside the wheal and the pricks in which a guineapig failed to react out of the six were recorded as no-pain responses.The pinpricks were applied in the order of left, center, right, upper,center and lower sections inside the wheal at an interval of 3-5 secbetween the pricks. Prior to the injections, all animals were checkedfor their vocalization reaction to pinpricks as baseline responses. FIG.3 illustrates the local anesthetic efficacy in percent no-pain responseover time for both formulations. The Control animals exhibited 100%analgesic/anesthetic effect at 15 minutes after the injection. However,such effect disappeared quickly with about 50% activity remained after 1hour and virtually no activity after 4 hours. This is consistent withthe short-term anesthetic nature of lidocaine. F-20 provided prolongedlocal drug activity with about 50% and 40% analgesic activity observedat 12 and 24 hours, respectively, after the injection.

Example 19 Injectability Test for an Aqueous Gel Containing Insulin(F-43)

An aqueous gel coded as F-43 in the following composition was preparedusing the method as described in EXAMPLE 2, where the insulin was addedafter the microfluization step and the average diameter of particles inthe nanodispersion was about 40 nm.

Component % wt Recombinant Human Insulin 100 IU/g (or about 0.38% wt)POPC 30 Sesame oil 8 Cholesterol 1.2 Vitamin E succinate 0.6 Sucrose 2Glycerin 1.6 Meta-cresol 0.027 Phenol 0.16 Dibasic sodium phosphateanhydrous 0.065 L-methionine 0.15 Zinc oxide 0.378 Protamine base 0.027Water for Injection, USP, q.s. About 56

F-43 was a translucent one-phase gel. The injectability of F-43 wasdetermined against the Acceptable Injectability Criterion. The maximumforce required during the injectability test is recorded as the mostrelevant measurement parameter for injectability. F-43 was filled into alcc B-D syringe (B-D Luer-Lok Tip, ref 309628) to which a ½″ long 25 Gneedle (EXEL, Hypodermic needle, ref 26403) was attached. The filledsyringe was loaded onto a syringe pump to which a force meter (AdvancedPrecision Instrument Model HP-500) was attached against the plunger endto measure to force applied to extrude the syringe contents. The syringepump was set at 2 cc/min speed and 0.4 mL extrusion volume. The forcewas recorded in pounds. In the “push” mode, the force is recorded asnegative. A representative injection force versus time profile for F-43is shown in FIG. 6 (upper panel). For comparison, the force profile fromthe same composition as F-43, but prepared by one-step vigoroushomogenization of all the components together (“Same composition byother method”). With a maximal injection force of less than 1.5 pounds,F-43 is regarded as highly injectable and meeting the AcceptableInjectability Criterion. It is compared very favorably to “Samecomposition by other method”, which required a maximum force of about 14pounds in the evaluation. F-43 was also filled into an insulin pencartridge (Eli Lily Humulin N Cartridge) and injected using a peninjector device (HumaPen Luxura by Eli Lilly and company). At a dialedinjection volume of 20 U, the injected volume in repeated injections wasfound to be accurate and precise. F-43 is well suited for pen injectors.

Example 20 Preparation of a Long-acting Depot Comprising an AnhydrousGel Containing Prednisone

An anhydrous gel depot is prepared to contain about 40 mg/mL prednisoneusing a composition comprising 50% soy lecithin, 40% sesame oil, and 6%ethanol using the method as follows:

-   -   a. Mix soy lecithin, water and sesame oil to form a primary        dispersion.    -   b. Remove water by lyophilization to less than 1%.    -   c. Add ethanol.    -   d. Mixing to obtain an anhydrous gel.    -   e. Pass the gel though a 0.45-micron filter for sterilization.        The resultant gel is a long-acting depot for treatment of        inflammation.

Example 21 Preparation of an Anhydrous Gel Containing Ibuprofen

An anhydrous gel depot is prepared to contain about 4 to 20% wtibuprofen using a composition and method as described in EXAMPLE 20. Theresultant gel is a long-acting depot for treatment of inflammation andpain.

Example 22 Preparation of a Long-acting Depot Comprising an AnhydrousGel Containing Clotrimazole

An anhydrous gel depot is prepared to contain about 40 mg/mLclotrimazole using a composition and method as described in EXAMPLE 20.The resultant gel is a long-acting depot for treatment of fungalinfection.

Example 23 Preparation of a Long-acting Depot Comprising an AnhydrousGel Containing Risperidone

An anhydrous gel depot is prepared to contain about 40 mg/mL Risperidoneusing a composition and method as described in EXAMPLE 6. The resultantgel is a long-acting depot for treatment of psychological disorder.

Example 24 Preparation of a Long-acting Depot Comprising an AnhydrousGel Containing Tamoxifen Citrate

An anhydrous gel depot is prepared to contain about 51 mg/mL TamoxifenCitrate using a composition and method as described in EXAMPLE 6. Theresultant gel is a long-acting depot for treatment of cancer.

Example 25 Preparation of a Long-acting Depot Comprising an AnhydrousGel Containing Diazepam

An anhydrous gel depot is prepared to contain about 40 mg/mL Diazepamusing a composition and method as described in EXAMPLE 20. The resultantgel is a long-acting depot for treatment of anxiety.

Example 26 Preparation of a Long-acting Depot Comprising an AnhydrousGel Containing Docetaxel

An anhydrous gel depot is prepared to contain about 40 mg/mL Docetaxelusing a composition and method as described in EXAMPLE 20. The resultantgel is a long-acting depot for treatment of cancer.

Example 27 Prolonged (24 hr) Insulin Pharmacodynamic Effect by AqueousGels Containing Insulin (F-43 and F-44)

The objective of this study was to evaluate two aqueous gel PGcompositions containing recombinant human insulin F-43 (as in EXAMPLE19) and F-44 in the streptozotosin (STZ)-induced type-I diabetic SpragueDawley rat model. This study compared the pharmacodynamic or PD (i.e.,blood glucose level versus time) profiles for two PG depots with the PDprofiles for two marketed basal insulin drugs (Insulin NPH or Humulin Nand Lantus) all at about 100 IU/mL strength. F-44 was prepared as anAqueous gel comprising 100 IU/mL recombinant human insulin, 50% POPC,zinc, protamine, phenol, m-cresol, glycerol, sodium phosphate and about40% water using the method as described in EXAMPLE 2, wherein theinsulin was added after the microfluization step.

Type I diabetes in Sprague Dawley rats was induced by intravenousinjection of STZ. Twenty-four (24) STZ-treated rats were randomlydivided into 4 groups. Small blood samples were taken from the lateraltail vein of each animal and the blood glucose levels were measuredusing a glucometer. The animals were successfully assigned to treatmentgroups such that there were no significant differences between groups inbody weight or blood glucose level as measured by one-way ANOVA(P>0.05). The animals were fasted for at least 12 hours and then theblood glucose levels were measured just prior to administration of thetest articles. The four formulations were administered subcutaneously at20 IU/kg. Blood glucose levels were measured at −0.25, 0.25, 5, 1, 2, 4,6, 8 16 and 24 hr post-insulin administration in Experiment 1. Bloodglucose levels were focused on the late time points for experiment 2 at−0.25, 1, 2, 12, 14, 16, 18, 20, 22, 24 and 36 hr post-insulinadministration. The blood glucose data versus time was graphed and thedata was analyzed with a two-way ANOVA with Bonferroni's post hoc testto evaluate pair wise comparisons between test formulations at eachmeasurement time point. Experiment 1 indicated that treatment with PGformulated insulin resulted in 24 hours of good glycemic control (bloodglucose level maintained between 50 and 130 mg/dl) for F-44 and about 18hours for F-43 (FIG. 7). The duration of glycemic control ranking was:F-44 (>24 hr) >F-43 (18 hr) >Humulin N (˜12-24 hr) >Lantus (12 hr).

All four tested formulations exhibited similar onset of action byachieving glycemic control within about 1 hour after the subcutaneousinjection. F-44 exhibited significantly lower blood glucose at latertime points (between 16 to 24 hrs) compared to the three other formulas.

There was no significant blood glucose level difference detected atearly time points. In addition, F-43 was able to keep blood glucosecontrolled for up to 18 hours after dosing. A repeated study (Experiment2) also confirmed the findings from Experiment 1; that is, F-44 cancontrol blood glucose up to 24 hrs and F-43 can maintain low bloodglucose level (<130 mg/dl) for at least 18 hrs. In comparison,Lantus-treated rats achieved glycemic control for only 12 hours. HumulinN showed the highest animal-to-animal variability in blood glucoselevel.

Example 28 Prolonged (24 hr) and Peak-less Pharmacokinetic Profile ofHuman Insulin Following Subcutaneous Injection of an Aqueous GelContaining Human Insulin (F-43)

The objective of this study was to evaluate F-43 (EXAMPLES 19 and 26) inSTZ-induced type-I diabetes conscious Sprague Dawley rats by comparingPK profiles (plasma insulin versus time) with a marketed insulin drug(NPH Insulin/Humulin N).

Type I diabetes in Sprague Dawley rats was induced by intravenousinjection of STZ. Eight (8) rats (all having STZ-induced type Idiabetes, Sprague Dawley) were placed into two treatment groups (F-43and NPH). Small amounts of blood samples were taken from the lateraltail vein of each animal and the blood glucose levels were measuredusing a glucometer. 0.3 ml of whole blood was taken from jugular veinand placed in EDTA-coated tubes for plasma separation. Theconcentrations of human insulin in plasma were measured using humaninsulin ELISA kit and RIA kits. The animals were assigned to treatmentgroups such that there were no significant differences between groups inbody weight or blood glucose level as measured by two-tailed student ttest (P>0.05). The animals were fasted for at least 12 hours and thenthe blood glucose levels were measured just prior to administration ofthe test formula.

The two formulations were administered subcutaneously at 20 IU/kg. Bloodglucose levels were measured at pre, 1, 2, 4, 6, 8, 16, 20 and 24 hrpost-insulin administration. Blood glucose and insulin levels versustime was graphed and the data was analyzed with a two-way ANOVA withBonferroni's post hoc test to evaluate pair wise comparisons betweentest formulations at each measurement time point. The data indicatedthat treatment resulted in about 24 hr glycemic control (blood glucoselevel maintained at below 130 mg/dl) by F-43 or about 16 hr byNPH/Humulin N (FIG. 8B -lower panel). The rats treated with F-43exhibited a steady and prolonged plasma insulin concentration profilebetween about 300 to 400 μIU/mL by the ELISA or between 250 and 400μIU/mL by the RIA method for about 24 hours, whereas the NPH-treatedrats showed continuous drop in plasma insulin concentration, whichdiminished at about 24 hours. Moreover, treatment with F-43 resulted ina “peak-less” PK profile (FIG. 8A—upper panel). Both tested formulationsexhibited similar onset of action, achieving glycemic control withinabout the first hour after the SC administration.

Example 29 Single-phase and Content Uniformity Study of F-43

The objective of this study was to demonstrate the single-phasestability and the concentration uniformity of insulin in F-43 (as inEXAMPLE 19) as studied using an insulin pen-injector.

Procedure: A) Day 1:

-   -   1. Insert one pen injector cartridge vial filled with F-43 or        Humulin N (NPH) into a pen injector (HumaPen Luxura by Eli Lilly        and Company).    -   2. Roll the pen back and forth 10 times and turn the pen up and        down 10 times to mix the content in the cartridge vial.    -   3. Attach a Novofine 28 G×12 mm needle.    -   4. Turn dose knob to 20 units and inject the content into a        small plastic vial, record the weight of the content injected.        Repeat the injection 9 times.        Determine concentration of insulin in each injection by HPLC        analysis.

B) Day 2,7 and 14:

Repeat the Day 1 procedure except Step 2.

Results Insulin IU/g Insulin IU/g Sample ID Day 1 Day 2 Sample ID Day 1Day 2 Day 7 Day 14 Humulin N, Inj #1 91.8 296.9 F43, Inj #1 104.1 100.6113.8 97.1 Humulin N, Inj #2 95.4 265.5 F43, Inj #2 105.1 98.7 110.9 96Humulin N, Inj #3 98.8 220.2 F43, Inj #3 104.1 100.2 97.6 95.9 HumulinN, Inj #4 96.4 167.1 F43, Inj #4 101.8 102.8 98.1 97.3 Humulin N, Inj #5101.9 110.4 F43, Inj #5 100.9 104.9 96.6 97 Humulin N, Inj #6 111.6 74.9F43, Inj #6 99.6 102.5 95.4 96.7 Humulin N, Inj #7 109.4 51.4 F43, Inj#7 102.6 103.7 98.2 96.6 Humulin N, Inj #8 110 26.1 F43, Inj #8 103.1103.5 100.1 95.9 Humulin N, Inj #9 89.5 15.8 F43, Inj #9 103.4 104.497.4 95.6 Humulin N, Inj #10 90.6 21.3 F43, Inj #10 103.7 104.2 97.597.7 Avg 99.5 124.95 Avg 102.84 102.55 100.57 96.56 RSD 8.3 105.8 RSD1.7 2.1 6.4 0.7 CV 8.4 84.7 CV 1.6 2.0 6.3 0.7

Conclusion: F-43 exhibited good content uniformity over time evenwithout implementing the pre-dosing mixing ritual that is required forHumulin N. Humulin N on the other hand showed highinjection-to-injection variability between about 8% and 85%. A separatestudy also shown that the F-43 remained uniform in its insulin contentafter centrifugation at no less than 1000 RPM for no less than 5minutes.

Example 30 Preparation of an Aqueous Gel Containing Insulin Determir forBasal Insulin Therapy

The Example is directed to preparation of an aqueous gel containinginsulin detemir, which is an insulin analog originally created by NovoNordisk. An aqueous gel is prepared in a composition similar to F-43 asin EXAMPLE 19, wherein the Recombinant Human Insulin is replaced with100 to 400 IU/mL insulin determir and the other components include POPC,sesame oil, sucrose, m-cresol, phenol, methionine and water. The methoddescribed in EXAMPLE 2 is used wherein insulin determir, is added beforeor after the nanodispersion is formed. This composition is intended forbasal insulin therapy to provide glycemic control for 24 hours orlonger.

Example 31 Preparation of an Aqueous Gel Containing NPH Insulin forBasal Insulin Therapy

The Example is directed to preparation of an aqueous gel containing NPHInsulin, which is also known as insulin isophane. An aqueous gel isprepared in a composition similar to F-43 as in EXAMPLE 19, wherein theRecombinant Human Insulin is replaced with 100 to 400 IU/mL NPH insulinand the other components include POPC, sesame oil, sucrose, glycerol,m-cresol, phenol, methionine, sodium phosphate and water. The methoddescribed in EXAMPLE 2 is used wherein NPH is added after thenanodispersion is formed. This composition is intended for basal insulintherapy to provide glycemic control for 24 hours or longer.

Example 32 Preparation of an Aqueous Gel Containing botulism toxin typeA for an Aesthetic Medicine Indication

The Example is directed to preparation of an aqueous gel in acomposition similar to F-43 as in EXAMPLE 19, wherein the RecombinantHuman Insulin is replaced with 100 units/g (about 0.5 nanograms/g) ofBOTOX® or purified botulinum toxin type A. The method described inEXAMPLE 2 is used wherein purified botulinum toxin type A is addedbefore or after the nanodispersion is formed. This composition isintended for the temporary improvement in the appearance of moderate tosevere glabellar lines associated with corrugator and/or procerus muscleactivity in adult patients ≦65 years of age.

Example 33 PG Structural Characterization by Small Angle X-RrayScattering (SAXS)

Small-angle X-ray scattering (SAXS) is an analytical technique thatprovides nanoscale information of particle or lattice systems in termsof such parameters as averaged particle sizes, shapes, distribution, andspacing etc. This information can be used to quantitative determine thestructural organization of materials such as gels. For a comparison oftwo gel samples, SAXS can show either differences in structures(particle size, stacking etc.) or the degree of molecular order whichdifferentiates an ordered structure from a less ordered or randomstructures. SAXS data is usually presented in diffractograms (FIG. 10).Peaks observed in a SAXS diffractogram is measured for scattering angle(on X-axis) which corresponds to lattice spacing in an ordered structureand the scattering intensity (peak height) and sharpness, which relateto degree of order.

Two gel samples containing insulin were prepared and tested by SAXS. Thefirst one is an aqueous gel of the present invention in the F-43composition as described in EXAMPLE 19 and the other one contains thesame components as in F-43 but was prepared by combining all componentsand homogenizing extensively using a high-speed mixer (Minibeadbeater).The second composition (“F-43 by Direct Mixing”) was not prepared usingthe invention method.

The SAXS data were collected in a helium chamber using a Bruker M18XHFrotating anode generator operating at 50 kV and 50 mA supplying a Cu Kα(λ=1.541838 Å) radiation beam that was collimated using a pinholecollimator. Kβ radiation was filtered out with a Ni filter. A Highstarmultiwire detector was used to collect the data. The samples were loadedwithout modification into 0.9 mm borosilicate glass capillaries andsealed with epoxy. The samples were mounted in the He chamber on anautomated goniometer at sample to detector distance of 64.55 cm. Toprevent scatter from air He gas was purged into the chamber for 1 hourand then each sample was collected for 7200 seconds. The data weresmoothed and integrated over the 360° χ circle from 0.8 to 4.7° 20 in0.02 degree widths.

FIG. 10 shows diffractograms for both F-43 and F-43 by Direct Mixing.Each sample showed two peaks. Provided below are the lattice d spacing(d(Å)) calculated by assuming n=1 for the Bragg equation (nλ=2d sin (0))and degree of order or size of “crystalline” domains based on theScherrer equation:

$\left( {\tau = \frac{K\; \lambda}{\beta \; \cos \; \theta}} \right.$

Degree of Order Sample Peak # d (Å) (Scherrer Crystalline domain size(nm)) F-43 by 1 70.00 42.3 (3) Direct Mixing 2 34.70 16.0 (3) F-43 167.38 81.3 (5) 2 33.61 33.4 (5)

The SAXS data suggested that both samples have two phases (peaks).Compared to F-43 by Direct Mixing, F-43 exhibits somewhat differentlattice d spacing in phase 1 (peak 1) and a greatly increased ScherrerCrystalline domain size or degree of order in both phases. This can bealso seen in the sharpening of the peaks in F-43 as compared to F-43 byDirect Mixing.

This SAXS study shows that F-43 PG material produced by the method ofthis invention has an ordered structure and higher degree of structuralorder than the same composition prepared another method. The orderedstructure found in the PG of the present invention is consistent to thestacked balloon model as depicted in FIG. 9 and is believed to be thereason for the surprisingly good injectability of the PGs.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other documents.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional 10 features,modification and variation of the inventions embodied therein hereindisclosed may be resorted to by those skilled in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each ofthe narrower species and sub generic groupings falling within thegeneric disclosure also form 15 part of the invention. This includes thegeneric description of the invention with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

In addition, where features or aspects of the invention are described interms of Markush groups, those skilled in the art will recognize thatthe invention is also thereby described in terms of any individualmember or subgroup of members of the Markush group. Other embodimentsare set forth within the following claims.

What is claimed is:
 1. A nanoemulsion based injectable one-phase gelcomposition, comprising: about 20% to about 60% by weight of aphospholipid based on the total weight of the gel, wherein thephospholipid is a member selected from the group consisting of alecithin, phosphatidylcholine or a mixture thereof; 0.1% to 65% byweight water based on the total weight of the gel; 1% to 50% by weightof an oil based on the total weight of the gel, wherein the phospholipidcomprising particles are stacked together and have a size of less than200 nm in diameter; wherein the composition has an increased degree oforder as shown by an increase in the Scherrer crystalline domain sizeover a composition containing the same quantities of the same componentsbut prepared by a process without forming a nanodispersion and thediscrete phospholipid particles; and is extrudable or injectable througha 25 G, ½ inch long needle from a 1 cc syringe at an extrusion rate of 2cc/min by an applied force of no more than 12 pounds.
 2. The gelcomposition of claim 1, further comprising a pharmacologically activeagent having concentration of no more than 20% by weight of the finalgel composition.
 3. The gel composition of claim 2, wherein thepharmacologically active agent is a heat-sensitive pharmacologicallyactive agent.
 4. The gel composition of claim 2, wherein thepharmacologically active agent is a protein or peptide.
 5. The gelcomposition of claim 2, wherein the pharmacologically active agent is amember selected from the group consisting of an insulin, an insulinanalog, a crystalline insulin with zinc and/or protamine, an NPHinsulin, and a combination thereof.
 6. The gel composition of claim 1,wherein the phospholipid comprises 20% to 40% by weight based on thetotal weight of the gel.
 7. The gel composition of claim 1, wherein thephospholipid is a lecithin.
 8. The gel composition of claim 1, whereinthe oil is selected from the group consisting of a synthetic oil, avegetable oil, a medium chain oil, ethyl oleate, fatty acid, vitamin E,vitamin E succinate, cholesterol, or a mixture thereof.
 9. The gelcomposition of claim 1, further comprising one or more sugars selectedfrom the group consisting of sucrose, dextrose, lactose, glucose,trehalose, maltose, mannitol, sorbitol, glycerol, amylose, starch,amylopectin, or a mixture thereof.
 10. The gel composition of claim 1,further comprising one or more non-aqueous solvents selected from thegroup consisting of ethanol, propylene glycol, glycerol, sorbitol,polyethylene glycol, ethyl oleate, or a mixture thereof.
 11. The gelcomposition of claim 1, further comprising a functional pharmaceuticalexcipient selected from the group consisting of an acidifying agent, analkalizing agent, a pH buffering agent, a metal ion chelator, anantioxidant, a preservative, a tonicity/osmotic pressure modifier, acondensing agent, a solubilizing agent, or a mixture thereof.
 12. Thegel composition of claim 1, wherein the gel composition has asmall-angle X-ray scattering diffractogram of FIG. 10 and a lattice dspacing of 67 Å and 33 Å.